Epifluorescence Video Microscopy Research Articles

In vitro and in vivo testing of a novel local nicardipine delivery system to the brain: a preclinical study.

The management of patients with aneurysmal subarachnoid hemorrhage (aSAH) remains a highly demanding challenge in critical care medicine. Despite all efforts, the calcium channel antagonist nimodipine remains the only drug approved for improving outcomes after aSAH. However, in its current form of application, it provides less than optimal efficacy and causes dose-limiting hypotension in a substantial number of patients. Here, the authors tested in vitro the release dynamics of a novel formulation of the calcium channel blocker nicardipine and in vivo local tolerance and tissue reaction using a chronic cranial window model in mice. To characterize the release kinetics in vitro, dissolution experiments were performed using artificial cerebrospinal fluid over a time period of 21 days. The excipients used in this formulation (NicaPlant) for sustained nicardipine release are a mixture of two completely degradable polymers. A chronic cranial window in C57BL/6 mice was prepared, and NicaPlant slices were placed in proximity to the exposed cerebral vasculature. Epifluorescence video microscopy was performed right after implantation and on days 3 and 7 after surgery. Vessel diameter of the arteries and veins, vessel permeability, vessel configuration, and leukocyte-endothelial cell interaction were quantified by computer-assisted analysis. Immunofluorescence staining was performed to analyze inflammatory reactions and neuronal alterations. In vitro the nicardipine release profile showed an almost linear curve with about 80% release at day 15 and full release at day 21. In vivo epifluorescence video microscopy showed a significantly higher arterial vessel diameter in the NicaPlant group due to vessel dilatation (21.6 ± 2.6 µm vs 17.8 ± 1.5 µm in controls, p < 0.01) confirming vasoactivity of the implant, whereas the venous diameter was not affected. Vessel dilatation did not have any influence on the vessel permeability measured by contrast extravasation of the fluorescent dye in epifluorescence microscopy. Further, an increased leukocyte-endothelial cell interaction due to the implant could not be detected. Histological analysis did not show any microglial activation or accumulation. No structural neuronal changes were observed. NicaPlant provides continuous in vitro release of nicardipine over a 3-week observation period. In vivo testing confirmed vasoactivity and lack of toxicity. The local application of this novel nicardipine delivery system to the subarachnoid space is a promising tool to improve patient outcomes while avoiding systemic side effects.

Recombinant FVIII and FVIIa Added to Flowing Hemophiliac Blood Co-Localize with Platelet Aggregates Adherent to a Collagen Surface, Promoting Local Fibrin Deposition in Wake-like Patterns Around the Aggregates

Mural platelet thrombi are considered to facilitate hemostasis in hemophilia via rFVIII/ rFVIIa binding to platelet membranes with enhancement of local fibrin deposition. We therefore sought to show that Factors VIII and VIIa co-localize to platelet membranes of adherent platelet aggregates and promote local deposition of fibrin. We collected blood from 9 patients with severe hemophilia A just prior to a prophylactic dose of rFVIII, and from 8 normals, into a plastic tube containing 5 U/ml FC low MW heparin, 1.75 µg/ml of the Tab (anti-CD41) mo antibody, and 1.0 μg/ml of ALEXA 555-conjugated rabbit anti-mouse secondary antibody, plus 0, 11, 33 or 100 ng/ml rFVIII-Oregon green. Similar approaches were used for rFVIIa or fibrin. Fibrin was detected using the monoclonal antibody 59D8 directed against human β fibrin, together with the above Alexa555-conjugated rabbit anti-mouse secondary antibody, and co-localized to platelet aggregate surfaces using 10 µM quinicrine dihydrochloride. Blood at 37° C was withdrawn at 270 sec-1 through a flow chamber, over 150 µm glass cover slips with preadsorbed microfibrillar collagen, using epifluorescence video microscopy and digital image analysis (MetaMorph Premier software). Added rFVIII or rFVIIa co-localized (Fig. 1) with platelet membranes and increased with rFVIII concentration. With hemophiliac blood and 100 ng/ml rFVIII, percent co-localization at 7 min was 52.6 ± 7.7 (N=4). Platelet aggregate growth rate, image pixels/min from time 3 to 9 min, was 26% lower in hemophiliac blood, but increased (p = 0.0001) 2.34 ± 0.62 (6)-fold with rFVIII, and (p = 0.0005) 1.91 ± 0.56 (6)-fold with 30 nM rFVIIa (Fig. 2). These increases essentially restored (or only slightly augmented) platelet deposition to levels observed in normal blood. In contrast, the effect on local fibrin deposition was profound: fibrin deposition was delayed by 3-5 min in normal blood, but by >13 min in hemophiliac blood. Addition of rFVIII or rFVIIa shortened the latter time to 7-9 min. Increases in platelet aggregate growth rate, following a lag phase in growth rate, paralleled the onset of fibrin formation. Fibrin appeared as comet-shaped deposits (comet head 20-25 µm in diameter) centered over each platelet aggregate (3-10 µm), the comet tail trailing 60-75 µm downstream (Fig. 3). In flowing whole blood in real time, these data show that rFVIII and rFVIIa co-localize with platelet membranes of activated, surface-adherent platelet aggregates, and support fibrin deposition in the slow-flow wakes immediately surrounding the platelet aggregates. [Display omitted] [Display omitted] [Display omitted] DisclosuresGrabowski:CSL Behring: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk, Denmark: Research Funding. Gunnarsson:Novo Nordisk: Employment. Ezban:Novo Nordisk: Employment.

Visualization of coral host–pathogen interactions using a stable GFP-labeled Vibrio coralliilyticus strain

The bacterium Vibrio coralliilyticus has been implicated as the causative agent of coral tissue loss diseases (collectively known as white syndromes) at sites across the Indo-Pacific and represents an emerging model pathogen for understanding the mechanisms linking bacterial infection and coral disease. In this study, we used a mini-Tn7 transposon delivery system to chromosomally label a strain of V. coralliilyticus isolated from a white syndrome disease lesion with a green fluorescent protein gene (GFP). We then tested the utility of this modified strain as a research tool for studies of coral host–pathogen interactions. A suite of biochemical assays and experimental infection trials in a range of model organisms confirmed that insertion of the GFP gene did not interfere with the labeled strain’s virulence. Using epifluorescence video microscopy, the GFP-labeled strain could be reliably distinguished from non-labeled bacteria present in the coral holobiont, and the pathogen’s interactions with the coral host could be visualized in real time. This study demonstrates that chromosomal GFP labeling is a useful technique for visualization and tracking of coral pathogens and provides a novel tool to investigate the role of V. coralliilyticus in coral disease pathogenesis.

The role of calcium in apoptosis induced by 7β‐hydroxycholesterol and cholesterol‐5β,6β‐epoxide

Oxysterols, such as 7beta-hydroxy-cholesterol (7beta-OH) and cholesterol-5beta,6beta-epoxide (beta-epoxide), may have a central role in promoting atherogenesis. This is thought to be predominantly due to their ability to induce apoptosis in cells of the vascular wall and in monocytes/macrophages. Although there has been extensive research regarding the mechanisms through which oxysterols induce apoptosis, much remains to be clarified. Given that experimental evidence has long associated alterations of calcium (Ca(2+)) homeostasis to apoptotic cell death, the aim of the present study was to determine the influence of intracellular Ca(2+) changes on apoptosis induced by 7beta-OH and beta-epoxide. Ca(2+) responses in differentiated U937 cells were assessed by epifluorescence video microscopy, using the ratiometric dye fura-2. Over 15-min exposure of differentiated U937 cells to 30 muM of 7beta-OH induced a slow but significant rise in fura-2 ratio. The Ca(2+) channel blocker nifedipine and the chelating agent EGTA blocked the increase in cytoplasmic Ca(2+). Moreover, dihydropyridine (DHP) binding sites identified with BODIPY-FLX-DHP were blocked following pretreatment with nifedipine, indicating that the influx of Ca(2+) occurred through L-type channels. However, following long-term incubation with 7beta-OH, elevated levels of cytoplasmic Ca(2+) were not maintained and nifedipine did not provide protection against apoptotic cell death. Our results indicate that the increase in Ca(2+) may be an initial trigger of 7beta-OH-induced apoptosis, but following chronic exposure to the oxysterol, the influence of Ca(2+) on apoptotic cell death appears to be less significant. In contrast, Ca(2+) did not appear to be involved in beta-epoxide-induced apoptosis.

In vivo Imaging of Cutaneous T-Cell Lymphoma Migration to the Skin

Cutaneous T-cell lymphoma (CTCL) is characterized by the accumulation of malignant CD4(+) T cells in the skin. Although the expression of adhesion molecules and chemokine receptors on CTCL cells has been studied extensively on ex vivo isolated cells, very little is known about the dynamics and mechanisms of CTCL trafficking in vivo. However, detailed knowledge of the molecular cues mediating CTCL migration may be used to interfere with their homing to the skin. We made use of real-time intravital epifluorescence video and two-photon microscopy to visualize malignant T cells from Sezary syndrome (SS), a leukemic variant of CTCL, in dermal microvessels in mouse ear skin. We found that SS cells rolled along dermal venules in a P-selectin- and E-selectin-dependent manner at ratios similar to CD4(+) memory T cells from normal donors. We furthermore show that the chemokine CCL17/TARC, but not CCL27/CTACK, was sufficient to induce the arrest of SS cells in the microvasculature. However, a combination of both chemokines was required to induce extravasation of SS cells. Together, our experiments delineate the molecular adhesion cascade operant in SS cell homing to the skin in vivo.

Diffusion of DNA in Nanoslits

We observed the free diffusion of individual 10 kbp, (1/2)λ (24.2 kbp), and λ (48.5 kbp) double-stranded deoxyribonucleic acid (DNA) molecules in fused-silica nanoslits with depths from 541 to 24 nm using epifluorescence video microscopy. Diffusivity, D, scaled with nanoslit depth, h, according to D ∝ hα, where α was 0.47 ± 0.05, 0.51 ± 0.06, and 0.47 ± 0.05 for 10 kbp, (1/2)λ, and λ DNA, respectively, in disagreement with the value of two-thirds predicted by blob theory. We observed no change in scaling behavior for h less than the persistence length of DNA, as predicted by reflecting rod theory. The scaling of D with DNA length, N, followed D ∝ N−1, indicating hydrodynamic screening in accordance with Rouse dynamics. Our results establish these scaling exponents for smaller h and greater DNA confinement than previous studies.

Stochastic Single-Molecule Videomicroscopy Methods To Measure Electrophoretic DNA Migration Modalities in Polymer Solutions above and below Entanglement

We have studied the effects of polymer molar mass and concentration on the electrophoretic migration modalities of individual molecules of DNA in LPA, HEC, and PEO solutions via epifluorescent videomicroscopy. While both transient entanglement coupling (TEC) and reptation have been studied in the past, the transition between them has not. Understanding this transition will allow for polymer network properties to be optimized to enhance the speed and resolution of DNA separations in microfluidic devices. Near the overlap threshold concentration, C*, TEC is the dominant observed mode of DNA migration, and the observation frequency of TEC increases with increasing polymer molar mass. As polymer concentration is increased, observed TEC events reduce to zero while DNA reptation events become the only detected mechanism. Individual DNA molecules undergoing both migration mechanisms were counted in solutions of varying polymer molar masses and concentrations and were plotted against a dimensionless polymer concentration, C/C*. The data for LPA reduce to form universal curves with a sharp increase in DNA reptation at approximately 6.5C*. Analogous transition concentrations for PEO and HEC were observed at 5C* and 3.5C*, respectively, reflecting the different physical properties of these polymers. This transition correlates closely with the polymer network entanglement concentration, Ce, as measured by rheological techniques. The electrophoretic mobility of lambda-DNA in LPA polymer solutions was also measured and shows how a balance can be struck between DNA resolution and separation speed by choosing the desired prevalence of DNA reptation.

A detection system for microfluidic chips based on epifluorescence videomicroscopy and its analytical potentials

An epifluorescence videomicroscopy system with a light-emitting diode as an excitation source and a charge-coupled device matrix as a radiation detector was developed and studied. The system was used for studying mass transfer in microchannels of single- and two-phase systems. It was shown that the developed detection system is suitable for studying mass transfer on a microchip. A one-dimensional physical and mathematical model was developed for mixing processes on a chip. A comparative analysis of experimental results and model concepts demonstrated the diffusion nature of the fluid flow mixing processes. Mass transfer in the two-phase water-octanol system was studied. Dye concentration profiles in a two-phase flow were recorded depending on the time of phase contact. The diffusion nature of mass transfer in the two-phase system was found.

Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p.

Exocytosis in the budding yeast Saccharomyces cerevisiae occurs at discrete domains of the plasma membrane. The protein complex that tethers incoming vesicles to sites of secretion is known as the exocyst. We have used photobleaching recovery experiments to characterize the dynamic behavior of the eight subunits that make up the exocyst. One subset (Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, and Exo84p) exhibits mobility similar to that of the vesicle-bound Rab family protein Sec4p, whereas Sec3p and Exo70p exhibit substantially more stability. Disruption of actin assembly abolishes the ability of the first subset of subunits to recover after photobleaching, whereas Sec3p and Exo70p are resistant. Immunogold electron microscopy and epifluorescence video microscopy indicate that all exocyst subunits, except for Sec3p, are associated with secretory vesicles as they arrive at exocytic sites. Assembly of the exocyst occurs when the first subset of subunits, delivered on vesicles, joins Sec3p and Exo70p on the plasma membrane. Exocyst assembly serves to both target and tether vesicles to sites of exocytosis.

Phase sensitive enhancement for biochemical detection using rotating paramagnetic particle chains

Paramagnetic particle suspensions placed in a rotating unidirectional magnetic field form magnetic chains that rotate with the same frequency as the field. The motion of the fluid and particles surrounding the chain differs in phase and frequency from the chain rotation, a phenomenon that forms the basis of a sensitive detection scheme. Fluorescent particles that bind to the paramagnetic particles through their surface chemistry are used to demonstrate the concept. Epifluorescence video microscopy is used to capture images of the rotating chains. View windows placed over sequential images of rotating chains allows for measurement of the fluorescence brightness in the window, which is composed of periodic signal from the steady rotation of the chain plus the background. A lock-in reference synchronized to the chain rotation is used to enhance the fluorescence signal from chain and improve signal to noise. Two different modes of chain rotation and signal collection are demonstrated. This technique can be used to develop a fast and sensitive, homogenous microdevice based solid-phase immunoassay.

Visualization of cell death in vivo during murine endotoxin-induced uveitis.

To develop a technology to image cell death in the eye of a live mouse and to apply that technology to characterize the role of apoptosis and necrosis in the evolution of endotoxin-induced uveitis (EIU), a standard model of intraocular inflammation. To induce EIU, 250 ng Escherichia coli 055:B5 lipopolysaccharide was injected into the vitreous body of BALB/c mice. At 0, 6, 10, 16, 20, 24, 48, 72, and 96 hours and on day 7 after endotoxin injection, annexin V and propidium iodide were injected into the anterior chamber of these mice, and labeled cells were observed by using intravital epifluorescence video microscopy. Iris and corneal wholemounts isolated from the mice were also evaluated with standard and confocal fluorescence microscopy. TUNEL staining was performed on iris wholemounts taken from additional mice similarly injected with endotoxin, to confirm the in vivo results. Uveitis was induced in all the mice that received an endotoxin injection. The percentages of annexin V(+) propidium(-) cells, annexin V(+) propidium(+) cells, and annexin V(-) propidium(+) cells in the iris tissues visible by intravital microscopy were comparable to those observed by TUNEL staining in vitro. In addition, intravital microscopy allowed observation of labeled cells in the aqueous humor and on the surface of the lens. Both the number and the pattern of labeled cells changed dramatically over time. The cells stained with annexin V had a variety of morphologies, including small and round, round with a lobulated or a kidney-shaped nucleus, dendriform, and irregular. A technique was developed to image cell death in the anterior segment of the eye in vivo and used to demonstrate that the number and proportion of early apoptotic (annexin V(+) propidium(-)) and late apoptotic or necrotic (annexin V(+) propidium(+) and annexin V(-) propidium(+)) cells change over the course of EIU. A variety of inflammatory cells and resident cells undergo apoptosis, or possibly necrosis, which may contribute to the rapid resolution of EIU. This in vivo technique will be a valuable tool for future studies on the resolution of ocular inflammation.

Continuous real time ex vivo epifluorescent video microscopy for the study of metastatic cancer cell interactions with microvascular endothelium.

Recent studies suggest that only endothelium-attached malignant cells are capable of giving rise to hematogenous cancer metastases. Moreover, tumor cell adhesion to microvascular endothelium could be crucial in metastasis predilection to specific organs or tissues. However, the existing in vitro and in vivo techniques do not provide for sufficient delineation of distinct stages of a dynamic multi-step intravascular adhesion process. Here we report the development of an experimental system allowing for prolonged continuous ex vivo real-time observation of malignant cell adhesive interactions with perfused microvessels of a target organ in the context of its original tissue. Specifically, the vasculature of excised dura mater perfused with prostate cancer cells is described. An advantage of this technique is that selected fluorescently labeled tumor cells can be followed along identified vascular trees across the entire tissue specimen. The techniques provide for superior microvessel visualization and allow for uninterrupted monitoring and video recording of subsequent adhesion events such as rolling, docking (initial reversible adhesion), locking (irreversible adhesion), and flattening of metastatic cancer cells within perfused microvasculature on a single cell level. The results of our experiments demonstrate that intravascular adhesion of cancer cells differs dramatically from such of the leukocytes. Within dura microvessels perfused at physiological rate, non-interacting, floating, tumor cells move at velocities averaging 7.2 x 10(3) microm/s. Some tumor cells, similarly to leukocytes, exhibit rolling-like motion patterns prior to engaging into more stable adhesive interactions. In contrast, other neoplastic cells became stably adhered without rolling showing a rapid reduction in velocity from 2 x 10(3) to 0 microm/s within fractions of a second. The experimental system described herein, while developed originally for studying prostate cancer cell interactions with porcine dura mater microvasculature, offers great flexibility in adhesion experiments design and is easily adapted for use with a variety of other tissues including human.

Involvement of Glycoprotein VI in Platelet Thrombus Formation on Both Collagen and von Willebrand Factor Surfaces Under Flow Conditions

We studied the role of glycoprotein (GP) VI in platelet adhesion and thrombus formation on the immobilized collagen and von Willebrand factor (vWF) surface under flow conditions. Whole blood obtained from 2 patients with GP VI-deficient platelets and the effects of the Fab of anti-GP VI antibody (Fab/anti-GP VI) were tested. Blood containing platelets rendered fluorescent by mepacrine was perfused on immobilized type I collagen or vWF under controlled wall shear rate. Platelet adhesion and thrombus formation were detected by epifluorescent videomicroscopy. The percentage of surface coverage by the platelets was calculated. Fc receptor gamma-chain and spleen tyrosine kinase (Syk) were immunoprecipitated from the lysate of platelets stimulated by vWF plus ristocetin and then analyzed by antiphosphotyrosine immunoblotting. No platelet attachment was seen on the surface of collagen even after 9 minutes of perfusion of blood at relatively low (100 s(-1)) or high (1500 s(-1)) wall shear rate, either in the case of blood containing GP VI-deficient platelets or in the presence of Fab/anti-GP VI, whereas significant platelet thrombus formation was noted after control blood perfusion. Such interference with the actions of GP VI also reduced firm platelet adhesion on immobilized vWF. vWF-induced tyrosine phosphorylation of GP VI-associated Fc receptor gamma-chain followed by Syk activation occurred in normal platelets, but little activation of Syk occurred in GP VI-deficient platelets. GP VI plays crucial roles in platelet thrombus formation on the surface of collagen under flow conditions in humans and is also involved in the process of firm platelet adhesion on the surface of vWF.

Functional significance of adenosine 5'-diphosphate receptor (P2Y(12)) in platelet activation initiated by binding of von Willebrand factor to platelet GP Ibalpha induced by conditions of high shear rate.

The role of the adenosine 5'-diphosphate receptor P2Y(12) in platelet activation initiated by the von Willebrand factor (VWF)-GP Ibalpha interaction under high shear rate was investigated. Blood samples were obtained from 11 donors. Shear-induced platelet aggregation was detected by optically modified cone-plate viscometer. Shear-induced VWF binding, P-selectin expression, and microparticle release were detected by flow cytometry. Platelet interaction with immobilized VWF was also investigated by parallel-plate flow chamber equipped with epifluorescent videomicroscopy. Effects of a selective P2Y(12) antagonist AR-C69931 MX were tested. AR-C69931 MX inhibited shear-induced platelet aggregation in a dose-dependent manner, achieving the maximum inhibition at 100 nmol/L. The extent of aggregation after exposure to a shear rate of 10 800 s(-1) for 6 minutes in the presence of 100 nmol/L AR-C69931 MX was 32.4+/-8.2% (mean+/-SD), which was significantly lower than the value in the controls of 69.7+/-9.6% (P<0.01). The inhibiting effects of AR-C69931 MX were reversed by exogenous addition of adenosine 5'-diphosphate. Shear-induced VWF binding and P-selectin surface translocation, which occurred in 4696+/-911 and 5964+/-784, respectively, of 10 000 measured platelets, was also inhibited by AR-C69931 MX (100 nmol/L) to 1948+/-528 and 2797+/-718, respectively (P=0.0018 and P=0.0009). Microparticle release was similarly inhibited. In a flow chamber experiment, firm platelet attachment on immobilized VWF was inhibited by AR-C69931 MX, whereas transient interaction was not influenced. All the above reactions were completely inhibited by blocking VWF-GP Ibalpha interaction. We have demonstrated that the stimulation of P2Y(12) is involved in platelet activation initiated by the binding of VWF to GP Ibalpha induced by a high shear rate.

Blockade of adenosine diphosphate receptors P2Y12 and P2Y1 is required to inhibit platelet aggregation in whole blood under flow

AbstractUsing heparinized whole blood and flow conditions, it was shown that adenosine 5′-diphosphate (ADP) receptors P2Y12 and P2Y1 are both important in direct shear-induced platelet aggregation and platelet aggregation subsequent to initial adhesion onto von Willebrand factor (vWf)–collagen. In the viscometer, whole blood was subjected to shear rates of 750, 1500, and 3000 s−1 for 30 seconds at room temperature. The extent of aggregation was determined by flow cytometry. The P2Y12antagonist AR-C69 931MX (ARMX) reduced shear-induced aggregation at these rates by 56%, 54%, and 16%, respectively, compared to control samples. Adenosine 3′,5′-diphosphate (A3P5P; P2Y1antagonist) inhibited shear-induced aggregation by 40%, 30% and 29%, respectively, compared to control samples. Blockade of both ADP receptors at 3000 s−1 with ARMX plus A3P5P further reduced the platelet aggregation by 41% compared to the addition of ARMX alone (57% compared to control samples). Using a parallel-plate flow chamber, whole blood was perfused over bovine collagen type 1 at a wall shear rate of 3000 s−1 for 60 seconds. Platelet deposition was quantified with epifluorescence video microscopy and digital image processing. Blockade of P2Y12 alone or blockade of P2Y1 alone did not reduce thrombus formation on vWf–collagen. In contrast, blockade of both P2Y12 and P2Y1 reduced platelet deposition by 72%. These results indicate that combinations of antagonists of the ADP receptors P2Y12 and P2Y1 are effective inhibitors of direct shear-induced platelet aggregation and of platelet aggregation subsequent to initial adhesion under flow conditions. Inhibitors of these pathways are potentially useful as antiarterial thrombotic agents.

Two distinct mechanisms are involved in stent thrombosis under flow conditions

The mechanism of stent thrombosis was evaluated by a flow chamber model. A Palmaz-Schatz stent was placed on glass surfaces coated with type I collagen in a parallel-plate flow chamber. Platelets were rendered fluorescent with mepacrine and whole blood was perfused through the chamber. Platelet thrombi formed around the implanted stents were continuously recorded by epi-fluorescent video-microscopy. Platelet thrombi appeared on the collagen surface within a few minutes in the absence of function blocking monoclonal antibodies. Antibody blocking vWF interaction with GP Ib f completely inhibited the platelet thrombi around the stent, but had no effect on platelet thrombus formation in the space between the stents and collagen, while both were inhibited by anti-GP IIb/IIIa. Our results suggest that two distinct mechanisms, one mediated by vWF interaction with GP Ib f occurring at high shear rates, and the other mediated exclusively by GP IIb/IIIa occurring at low shear rates, are involved in the process of platelet thrombus formation in the presence of stents.

Improved blood compatibility of DLC coated polymeric material.

There is currently an increasing interest in the use of DLC (diamond like carbon) films in biomedical applications. These investigations making use of DLC in the biomedical area indicate its attractive properties. In this study, we succeeded in depositing DLC on polymer substrates and found the best conditions and method for this application. We evaluated the blood compatibility of polycarbonate substrates coated by DLC (PC-DLC) under different conditions by using epifluorescent video microscopy (EVM) combined with a parallel plate flow chamber. Segmented polyurethane (SPU), which has been used to fabricate medical devices including an artificial heart, and proven to have acceptable blood compatibility, was compared with polycarbonate substrates coated with DLC film. The EVM system measured platelet adhesion on the surface of the DLC, by using whole human blood containing Mepacrine labeled platelets perfuse at a wall shear rate of 100 s(-1) at 1 min intervals for a period of 20 min. PC-DLC demonstrated that Tecoflex showed higher complement activation than PC-DLC. There were significant differences between the PC-DLC substrates. On the basis of these results, it is recommended for use as a coating material in implantable blood contacting devices such as artificial hearts, pacemakers, and other devices. This DLC seems to be a promising candidate for biomaterials applications and merits further investigation.

EVM (Epifluorescent Video Microscopy) を用いた光不透過材料の抗血栓性相対評価システムの開発

EVM (Epifluorescent Video Microscopy) を用いた光不透過材料の抗血栓性相対評価システムの開発

Electrohydrodynamic instabilities and segregation of polysaccharides in capillary polymer solution electrophoresis

During capillary electrophoresis of negatively charged polysaccharides in polymer solutions as sieving media, concentration fluctuations develop due to electrohydrodynamic instabilities caused by polarization of the polyelectrolytic chains. This leads to deviations from electroneutrality far beyond the Debye layer and segregation of the initially homogeneous sample solution into aggregated sample-rich domains as verified by epifluorescence videomicroscopy imaging. As a result, anomalous and irregular peak profiles are obtained impeding the characterization of such complex sample mixtures. This effect appears at an electric field strength threshold value that depends on the molecular weight of the solute polymer molecules, pH, type and concentration of the polymer solution sieving media, and buffering conditions. The magnitude increases with increasing field strength and amount of sample injected. The aggregation onset, as evaluated by the value of the threshold potential, is affected by the charge density of the sample polymer molecules and Debye screening effects and investigated through variation of pH and ionic strength, respectively. Exchange of a simple base buffer component for small and multiply charged organic bases markedly increases the electric field strength necessary to trigger the electrohydrodynamic instabilities. Ultimately, the threshold value could be increased more than seven times by addition of an oppositely charged aminodextran polymer, thereby decreasing the analysis time. © 1999 John Wiley & Sons, Inc. Biopoly 49: 515–524, 1999

Platelet-activating factor mediates ischemia-induced leukocyte-endothelial adherence in newborn pig brain.

The authors examined the involvement of platelet-activating factor (PAF) in mediating leukocyte adherence to brain postcapillary pial venules and altering blood-brain barrier (BBB) permeability during basal conditions and during reoxygenation after asphyxia in newborn piglets. Intravital epifluorescence videomicroscopy, closed cranial windows, and labeling of leukocytes with rhodamine 6G allowed us to obtain serial measurements of adherent leukocytes within postcapillary venules. Blood-brain barrier breakdown was determined by optical measures of cortical extravascular fluorescence intensity after intravenous sodium fluorescein. Superfusion of PAF over the cortex induced a dose-dependent increase in leukocyte adherence to cerebral venules and leakage of fluorescein; with 1 micromol/L PAF, the magnitude of adherence and BBB breakdown was similar to that seen during reoxygenation after 9 minutes of asphyxia. Both adherence and loss of BBB integrity resulting from either exogenous PAF or asphyxia-reoxygenation could be significantly attenuated by intravenous administration of WEB 2086, a PAF receptor antagonist. Window superfusion of superoxide dismutase with PAF attenuated PAF-induced increases in adherence and associated fluorescein leakage. These findings indicate that PAF exhibits proinflammatory effects in piglet brain and that PAF contributes to leukocyte adherence and BBB breakdown after cerebral ischemia. These PAF effects are mediated by increases in superoxide radical generation.