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At least 595 records · Page 33Linked to original sources
PubMed1997

Sodium channel distribution within the rabbit atrioventricular node as analysed by confocal microscopy.

1. Paired 20 microns thick sections of fresh frozen tissue taken from the frontal plane of the rabbit atrioventricular (AV) nodal region were processed for histology and immunohistochemistry. Confocal microscopy was used to image the distribution of sodium channels using IgG (R12) developed against a highly conserved sequence in the interdomain 3-4 region of cloned sodium channels. 2. In ventricular and atrial cells, sodium channel immunofluorescence was localized to lateral membranes and T-tubules. In the open AV node, levels of sodium channel immunofluorescence in the transitional cell zone and in the lower nodal cell tract were comparable to that found in the atrial and ventricular myocardium. 3. In the enclosed AV node a gradation of sodium channel immunofluorescence is present such that peripherally located circumferential transitional cells display high levels of immunofluorescence, comparable to that of atrial and ventricular myocardium, while centrally located midnodal cells display decreased levels of or no immunofluorescence. 4. In order to correlate the distribution of sodium channels with the distribution of gap junctions, we used IgG directed against the carboxyl terminus of connexin43 (CT-360). Ventricular cell immunofluorescence was localized primarily to the intercalated disk region, while in the AV node, the pattern of distribution was found to be similar to that of sodium channels. 5. The reduced levels of and/or absence of immunofluorescence in the midnodal cell region indicates a paucity of sodium channel and connexin43 protein expression in this region of the AV node that would favour slow impulse conduction.

Animals↗
PubMed2005

Intracellular trafficking of transferrin-conjugated liposome/DNA complexes by confocal microscopy.

Intracellular trafficking of transferrin-conjugated dimethyldioctadecyl-ammonium bromide liposome (T1-liposome)/DNA complexes in HeLa cells was studied using the double-labeled fluorescence technique and confocal microscopy. The size of the T1-liposome/DNA complex was about 367 nm in diameter and the zeta-potential of it at a 5:1 (w/w) ratio was almost neutral. The intracellular pathway of the T1-liposome/DNA complex, noted as green (FITC), red (rhodamine) or yellow (FITC + rhodamine) fluorescence, was elucidated from the plasma membrane to the endosome (or lysosome), and finally to the nucleus. The results of this study indicate that plasmid DNA enters into the nucleus not only as a free form but as an associated form complexed with T1-liposome. More interestingly, the T1-liposome undergoes a nuclear location in the form of ordered structures. This could be a very useful piece of information in designing a safe and advanced gene delivery system.

DNA↗
PubMed2001

Confocal microscopy study of the different patterns of 2-NBDG uptake in rabbit enterocytes in the apical and basal zone.

D-Glucose uptake in isolated rabbit enterocytes was studied using confocal microscopy and 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NDBG), a D-glucose fluorescent analogue, by analysing the fluorescence of apical and basal enterocyte zones. Under normal conditions, apical fluorescence was always higher than basal, presumably due to the location of the Na+-D-glucose cotransporter in the brush-border membrane. After blocking this transporter with phlorizin, apical and basal fluorescence values were similar. This suggests that both brush-border and basolateral membranes participate in phlorizin-insensitive D-glucose transport, since transport across only one membrane cannot explain the uniform overall fluorescence observed. Similarly, after inhibiting the Na+-D-glucose cotransporter by incubating the enterocytes in a medium containing 0.5 mM Na+, neither apical nor basal fluorescence predominated. In contrast, with 130.5 mM extracellular Na+, apical fluorescence was clearly higher than basal fluorescence. These results suggest that phlorizin-insensitive, Na+-independent 2-NDBG uptake occurred through both brush-border and basolateral membranes, probably via the glucose uniporters GLUT2 and GLUT5, suggesting that the latter is a D-glucose transporter.

4-Chloro-7-nitrobenzofurazan↗
PubMed2000

Xenobiotic transport across isolated brain microvessels studied by confocal microscopy.

To identify specific transporters that drive xenobiotics from central nervous system to blood, the accumulation of fluorescent drugs was studied in isolated capillaries from rat and pig brain using confocal microscopy and quantitative image analysis. Luminal accumulation of daunomycin and of fluorescent derivatives of cyclosporine A (CSA) and ivermectin was concentrative, specific, and energy-dependent (inhibition by NaCN). Transport was reduced by PSC 833, ivermectin, verapamil, CSA, and vanadate, but not by leukotriene C(4) (LTC(4)), indicating the involvement of P-glycoprotein. Luminal accumulation of the fluorescent organic anions sulforhodamine 101 and fluorescein methotrexate was also concentrative, specific, and energy-dependent. LTC(4), chlorodinitrobenzene, and vanadate reduced transport of these compounds, but PSC 833 and verapamil did not, indicating the involvement of a multidrug resistance-associated protein (Mrp). Immunostaining localized P-glycoprotein and Mrp2 to the luminal surface of the capillary endothelium and quantitative polymerase chain reaction showed Mrp1 and Mrp2 expression. Finally, the HIV protease inhibitors saquinavir and ritonavir were potent inhibitors of transport mediated by both P-glycoprotein and Mrp. These results validate a new method for studying drug transport in isolated brain capillaries and implicate both P-glycoprotein and one or more members of the Mrp family in drug transport from central nervous system to blood.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1995

Confocal microscopy for structure and real-time pharmacology in blood vessels.

The structure and composition of living small blood vessels can be studied in great detail in three dimensions using confocal microscopy. Individual cells and their components can be visualised by vital dyes for the nucleus, cytoplasm or extracellular space. Specific ligands can then localise individual components such as membrane receptors with great precision. Cell function is unaffected, allowing the study, in real time, of the changing relation and contribution to vascular contraction or dilatation of different cell types particularly smooth muscle, endothelium and adventitia. This allows not only visualisation but quantification, using image analysis software. These techniques will be of particular value in assessing the contribution of form to function in pathological situations such as vascular remodelling in hypertension.

Animals↗
PubMed2002

Validation of confocal microscopy through focusing for corneal sublayer pachymetry.

PURPOSE: To evaluate the accuracy and precision of confocal microscopy through focusing (CMTF) for corneal sublayer pachymetry. METHODS: A tandem scanning confocal microscope equipped with a nonapplanating contact objective was used to perform CMTF. The accuracy of CMTF measurements was evaluated using nine custom-made calibration contact lenses (PMMA) with varying thickness (300-600 microm) and radius of curvature (7.0-9.0 mm). The influence of immersion fluid stabilization and the consequence of prolonged corneal examination were assessed by performing CMTF in rabbits. Additionally, factors related to the instrumental setup and to sedation of experimental animals were examined. RESULTS: For all calibration contact lenses, the thickness measured by CMTF was within +/-1.0 microm of the certified value. Varying the target thickness or radius of curvature had no consistent impact on the high accuracy of CMTF. When performing CMTF in vivo, z-axis motion was readily identified by sampling and comparing both in- and out-scans. Apart from involuntary eye movements, z-axis motion was due to initial thinning of the immersion fluid with stabilization obtained after approximately 1.5 minutes. Continued confocal examination led to slight but significant swelling of both the stroma (0.5 microm/min) and epithelium (0.1 microm/min). CONCLUSIONS: CMTF is an accurate and precise technique for corneal sublayer pachymetry with concurrent display of cellular morphology. The precision of CMTF can be improved by allowing time for methylcellulose stabilization and by performing repeated two-way (in and out) scans to account for z-axis motion.

Air↗
PubMed1996

The cellular localization of the neuropeptides substance P, neurokinin A, calcitonin gene-related peptide and neuropeptide Y in guinea-pig vestibular sensory organs: a high-resolution confocal microscopy study.

Four neuropeptides, substance P, neurokinin A, calcitonin gene-related peptide and neuropeptide Y, were detected by radioimmunoassay in guinea-pig vestibular end-organs. High-resolution confocal microscopy visualization of immunofluorescence staining was used to determine the cellular localization of these peptides. Substance P- and neurokinin A-like immunoreactivities were found to co-exist in afferent fibers innervating the peripheral regions of both the utricular and ampullar sensory organs. The immunoreactivity was more concentrated in the distal ends of the calyceal-shaped nerve endings that innervate type I sensory cells. While in the guinea-pig, nerve calyces and type I cells are distributed in both the central and peripheral regions of the sensory epithelia, immunoreactive calyces were found only in the peripheral regions. Calcitonin gene-related peptide-like immunoreactivity was localized in small bouton endings situated at the level of the base of the hair cells. These boutons were in a position to make axosomatic contacts with type II sensory cells and axodendritic contacts with afferent nerve endings. Calcitonin gene-related peptide immunoreactivity co-existed with choline acetyltransferase immunoreactivity. The localization and shape of these boutons identified them as the axonal endings of efferent vestibular fibers. Neuropeptide Y-like immunoreactivity was not observed in the actual sensory epithelium but in the underlying connective tissue, where it was located in varicose fibers along blood vessels. The synaptic position of the tachykinins is clearly distinct from that of calcitonin gene-related peptide. This segregation distinguishes the vestibular end-organs from most peripheral tissues where these peptides are co-localized. The tachykinin-immunoreactive afferent fibers are postsynaptic to the hair cells. If, as in somatic sensory endings, these fibers can be triggered to release the neuropeptides by an axon reflex type of activation, then the tachykinins could interfere directly with the function of type I and type II vestibular hair cells. Calcitonin gene-related peptide co-exists with acetylcholine in the efferent axonal endings that are presynaptic to type II hair cells and to afferent fibers. Calcitonin gene-related peptide can thus interfere by direct synaptic action with type II hair cells only. It may also regulate the activity of the tachykinin-containing afferents.

Animals↗
PubMed1995

Novel insights into human intestinal epithelial cell proliferation in health and disease using confocal microscopy.

Measurement of intestinal epithelial cell proliferation has provided important information concerning tissue responses in neoplasia, enteropathy, and adaptation. This study reexamined current concepts regarding intestinal proliferation by using a novel confocal microscopical technique to map mitotic figures accurately within the intact three dimensional framework of the crypts of Lieberkühn. The ability of confocal microscopy to simultaneously measure crypt morphology and internal detail, without disrupting spatial cell arrangements, has provided important new data. These question the ability of existing methods to accurately measure and interpret proliferative changes in the gut. This work investigates intestinal proliferation in children with coeliac disease, a well defined hyperproliferative disorder, in comparison with the healthy intestine. These results show that crypt cell division occurs with an equal probability in health and disease. In addition, increased crypt cell production rates are largely caused by a change in crypt size rather than a change in cell cycle time or crypt growth fraction and, as such, alter our understanding of kinetic responses in gastrointestinal disease.

Celiac Disease↗
PubMed2005

Pilot study on the sensitivity and specificity of in vivo reflectance confocal microscopy in the diagnosis of allergic contact dermatitis.

The validity, reproducibility, and specificity of patch-testing in the diagnosis of allergic contact dermatitis (ACD) have repeatedly been addressed. In vivo reflectance confocal microscopy (RCM) has been used for real-time evaluation of the histopathologic features of ACD. This pilot study was designed to determine the sensitivity and specificity of RCM in diagnosing ACD in reference to patch-testing. Sixteen participants were patch tested with allergens and control substances. Clinical scoring, digital photography, and RCM evaluation were performed at 72 hours, and RCM images were subjected to blinded evaluation. RCM evaluation parameters included stratum corneum (SC) disruption, parakeratosis, stratum spinosum (SS) and stratum granulosum (SG) spongiosis, and exocytosis. Overall, there was high specificity for all RCM features, ranging from 95.8% to 100%. Sensitivity ranged from 51.9% to 96.3%. Significant parameters with high sensitivity and specificity included spongiosis and exocytosis at the level of SS. Logistic regression analysis was performed on significant variables; P values were determined by chi2 analysis. RCM is a promising noninvasive technology for the evaluation of ACD. SC changes are not helpful in the diagnosis of ACD, although the presence of SG spongiosis and SS spongiosis shows high sensitivity in diagnosing ACD. Larger sensitivity and specificity studies are needed and the identification of ACD has to be based on a defined diagnostic algorithm. A limitation of this study is the small sample size; larger sensitivity and specificity studies are needed to confirm these findings. In addition, individual allergens have to be subjected to further evaluations in order to demonstrate the applicability of our findings for other contact allergens. In that regard, RCM may be considered as an adjunctive tool, rather than a substitute, to clinical evaluation.

Adult↗
PubMed1999

Effect of Ca2+ agonists in the perfused liver: determination via laser scanning confocal microscopy.

Ca2+ is a critical intracellular second messenger, but few studies have examined Ca2+ signaling in whole organs. The amplitude and frequency of Ca2+ oscillations encode important cellular information. Using laser scanning confocal microscopy in the indo 1 acetoxymethyl ester dye-loaded rat liver, we investigated the effect of various Ca2+ agonists that act at distinct mechanistic sites on Ca2+ signaling. Perfusion with suprathreshold doses of arginine vasopressin (AVP) (2-20 nM) caused a single Ca2+ wave that originated in the pericentral vein region and spread centrifugally to the periportal area. Lower doses of AVP (0.2-2 nM) caused multiple Ca2+ waves and Ca2+ oscillations. Perfusion with ATP (1. 4-17.5 microM) caused rapid transient elevations in intracellular free Ca2+ concentration ([Ca2+]i) occurring in isolated hepatocytes or groups of hepatocytes throughout the lobule and were of shorter duration than those due to AVP. Also in contrast to AVP, there was no specific anatomic location within the hepatic lobule that was more susceptible to ATP. Thapsigargin and cyclopiazonic acid did not cause a Ca2+ wave but rather produced a uniform and fairly simultaneous increase in [Ca2+]i in all hepatocytes in the lobule. Perfusion with 14 microM ryanodine produced a single transient spike in [Ca2+]i in a small number (<2%) of hepatocytes. Dantrolene, an inhibitor of Ca2+ release, reduced the increased [Ca2+]i occurring after AVP. Insight into the mechanism of action of these Ca2+-active compounds on Ca2+ signaling in the intact liver is provided.

Adenosine Triphosphate↗
PubMed2001

Qualitative and quantitative analysis of monocyte transendothelial migration by confocal microscopy and three-dimensional image reconstruction.

A novel method for qualitative and quantitative analysis of monocyte transendothelial migration is described. By labeling monocytes and endothelial cells with different fluorophores, and utilizing confocal microscopy and three-dimensional image reconstruction, transmigrating monocytes were resolved and quantified within a subendothelial collagen gel. Comparison of monocyte migration across endothelial monolayers derived from human brain microvessels versus umbilical veins revealed diapedesis across brain endothelium to be significantly delayed. Inclusion of astrocytes within the subendothelial collagen gel resulted in the formation of an array of astrocytic processes that simulated the glia limitans surrounding brain microvessels in situ, thus yielding a more physiologic paradigm of the blood-brain barrier. By virtue of its unique capacity to provide information on the total number of migrating cells, this analytic approach overcomes significant caveats associated with sampling only aspects of the migration process. The potential adaptability of this method to computer-assisted analysis further enhances its prospective use in high-throughput screening.

Astrocytes↗
PubMed1992

Neuropathology in depth: the role of confocal microscopy.

This review surveys the rapidly increasing applications of confocal microscopy in neuropathology and related areas of experimental neurology. The ability of this new instrument to "optically section" thick samples has opened up to microscopic examination a wide range of previously difficult specimens. The basic operating principle, trading field of view for axial resolution, is illustrated with a simple schematic comparison of conventional and confocal microscopes. Images can be collected in several different modes, utilizing fluorescence, reflectance, or transmitted light. The majority of the applications in neuropathology to date have focused on immunocytochemical labeling with peroxidase or fluorescent markers. New methods of specimen preparation designed explicitly for the confocal microscope are just beginning to appear. The trend toward new techniques that fully exploit the superior performance of confocal microscopes is expected to accelerate, which will quickly establish the confocal microscope as a routine laboratory tool for the neuropathologist.

Animals↗
PubMed2004

Reflectance confocal microscopy may differentiate acute allergic and irritant contact dermatitis in vivo.

BACKGROUND: Acute irritant contact dermatitis (ICD) and allergic contact dermatitis (ACD) may be difficult to distinguish by clinical or histologic assessment. Reflectance confocal microscopy (RCM) enables real-time, high-resolution skin imaging in vivo. OBJECTIVE: We sought to image, characterize, and distinguish acute ACD and ICD in vivo. METHODS: Volunteers with ACD were patch tested with an allergen and the irritant, sodium lauryl sulfate. RCM imaging and transepidermal water loss measurements were performed at 24 and 72 hours. Biopsy specimens were correlated with RCM images. RESULTS: Spongiosis, epidermal inflammatory cell infiltrate, and vesicle formation were observed in ACD and ICD. Compared with ACD, ICD showed greater disruption of the stratum corneum, and more parakeratosis. There was a significantly greater increase in transepidermal water loss for ICD compared with ACD. CONCLUSION: RCM is a promising tool for dynamic, noninvasive assessment and may help to differentiate acute ACD and sodium lauryl sulfate-induced ICD.

Adult↗
PubMed2004

Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux.

Exposure to light can destroy the ability of a molecule to fluoresce. Such photobleaching limits the use of fluorescence and confocal microscopy in biological studies. Loss of fluorescence decreases the signal-to-noise ratio and so image resolution; it also prevents the acquisition of meaningful data late during repeated scanning (e.g. when collecting three-dimensional images). The aim of this work was to investigate the role of oxygen in the photobleaching of fluorophores bound to DNA in fixed cells, and to explore whether anoxia could minimize such bleaching. Anoxia significantly reduced bleaching rates and changed the order of reaction of both propidium iodide (an intercalator) and chromomycin A3 (a minor-groove binder) bound to DNA; it afforded the greatest protection at low photon fluxes. However, it had no effect on the bleaching of the green fluorescent protein (GFP) covalently attached to a histone and so bound to DNA, probably because the protein shielded the chromophore from oxygen. Bleaching of all three fluorophores depended on photon flux. Practical ways of minimizing bleaching were examined, and examples of three-dimensional images of DNA marked by propidium and GFP (collected under standard and optimized conditions) are presented.

Cells, Cultured↗
PubMed2004

Fluorescein-methotrexate transport in rat choroid plexus analyzed using confocal microscopy.

One function of the vertebrate choroid plexus (CP) is removal of potentially toxic metabolites and xenobiotics from cerebrospinal fluid (CSF) to blood for subsequent excretion in urine and bile. We have used confocal microscopy and quantitative image analysis to follow transport of the large organic anion fluorescein-methotrexate (FL-MTX) from bath (CSF side) to blood vessels in intact rat CP and found concentrative transport from CSF to blood. With 2 microM FL-MTX in the bath, steady-state fluorescence in the subepithelium and vascular spaces exceeded bath levels by 5- to 10-fold, but fluorescence in epithelial cells was below bath levels. FL-MTX accumulation in subepithelium and vascular spaces was reduced by NaCN, Na removal, and by other organic anions, e.g., MTX, probenecid, and estrone sulfate. Increasing medium K 10-fold had no effect. None of these treatments affected cellular accumulation. However, two observations indicated that apical FL-MTX uptake was indeed mediated: first, cellular accumulation was a saturable function of medium substrate concentration; and second, digoxin and MK-571 reduced FL-MTX accumulation in the subepithelial/vascular spaces but also increased cellular accumulation severalfold. In the presence of digoxin and MK-571, cellular accumulation was concentrative, specific, and Na dependent. Thus transepithelial FL-MTX transport involved the following two mediated steps: Na-dependent uptake at the apical membrane and electroneutral efflux at the basolateral membrane, possibly on Oatp2 and Mrp1.

Animals↗
PubMed2005

Osteoclast responses to lipopolysaccharide, parathyroid hormone and bisphosphonates in neonatal murine calvaria analyzed by laser scanning confocal microscopy.

Because the development and activity of osteoclasts in bone remodeling is critically dependent on cell-cell and cell-matrix interactions, we used laser confocal microscopy to study the response of osteoclasts to lipopolysaccharide (LPS; 10 microg/ml), parathyroid hormone (PTH; 10(-8) M), and bisphosphonates (BPs; 1-25 microM clodronate or 0.1-2.5 microM risedronate) in cultured neonatal calvaria. Following treatment with LPS or PTH (<48 hr), osteopontin (OPN) and the alphavbeta3 integrin were found colocalized with the actin ring in the sealing zone of actively resorbing osteoclasts. In contrast, non-resorbing osteoclasts in BP-treated cultures showed morphological abnormalities, including retraction of pseudopods and vacuolization of cytoplasm. In the combined presence of LPS and BP, bone-resorbing osteoclasts were smaller and the sealing zone diffuse, reflecting reduced actin, OPN, and beta3 integrin staining. Depth analyses of calvaria showed that the area of resorbed bone was filled with proliferating osteoblastic cells that stained for alkaline phosphatase, collagen type I, and bone sialoprotein, regardless of the presence of BPs. These studies show that confocal microscopy of neonatal calvaria in culture can be used to assess the cytological relationships between osteoclasts and osteoblastic cells in response to agents that regulate bone remodeling in situ, avoiding systemic effects that can compromise in vivo studies and artifacts associated with studies of isolated osteoclasts.

Acid Phosphatase↗
PubMed2005

Determination of microvessel permeability and tissue diffusion coefficient of solutes by laser scanning confocal microscopy.

Interstitium contains a matrix of fibrous molecules that creates considerable resistance to water and solutes in series with the microvessel wall. On the basis of our preliminary studies, by using laser-scanning confocal microscopy and a theoretical model for interstitial transport, we determined both microvessel solute permeability (P) and solute tissue diffusion coefficient (D) of alpha-lactalbumin (Stokes radius 2.01 nm) from the rate of tissue solute accumulation and the radial concentration gradient around individually perfused microvessel in frog mesentery. P(alpha-lactalbumin) is 1.7 +/- 0.7(SD) x 10(-6) cm/s (n = 6). D(t)/D(free) for alpha-lactalbumin is 27% +/- 5% (SD) (n = 6). This value of D(t)/D(free) is comparable to that for small solute sodium fluorescein (Stokes radius 0.45 nm), while p(alpha-lactalbumin) is only 3.4% of p(sodium fluorescein). Our results suggest that frog mesenteric tissue is much less selective to solutes than the microvessel wall.

Animals↗
PubMed2003

In vivo confocal microscopy for evaluation of wound healing following corneal refractive surgery.

Understanding of corneal wound healing plays an important role, not only in management of corneal infections, but especially in refractive surgery. A better control of wound healing mechanisms might improve the results of such resculpturing techniques and help to avoid complications arising from these procedures. While studies have been focused in different aspects of corneal wound healing, our knowledge has increased greatly during the last years. Many problems associated with corneal healing also contribute to clinical pathology following corneal surgery. Understanding of such conditions has been augmented by the continuously developing corneal imaging techniques. We have used in vivo confocal microscopy (IVCM) for assessing corneas subjected to refractive surgery as well as corneas with common complications resulting from such procedures. IVCM has become a powerful tool for examining corneal cells, nerves, inflammations and infections. It allows information to be acquired repeatedly and at subbiomicroscopic levels that earlier had been obtainable only by invasive microscopic methods. Pre-examining corneas preoperatively by IVCM in order to reveal diseases or conditions in which elective refractive surgical procedures should not be undertaken or to select the ideal operation technique may help to avoid complications in the future. Measurement of the thickness of corneal sublayers or estimation of the thickness of a laser in situ keratomileusis flap or wound bed are other applications in which confocal microscopy may be valuable. In this article we attempt to describe the in vivo confocal findings of common refractive procedures and their complications, and discuss their biology in light of the existing knowledge on wound healing phenomena.

Animals↗