Fluorescence Video Research Articles

Estrogen modulates neuronal movements within the developing preoptic area–anterior hypothalamus

The preoptic area-anterior hypothalamus (POA-AH) is characterized by sexually dimorphic features in a number of vertebrates and is a key region of the forebrain for regulating physiological responses and sexual behaviours. Using live-cell fluorescence video microscopy with organotypic brain slices, the current study examined sex differences in the movement characteristics of neurons expressing yellow fluorescent protein (YFP) driven by the Thy-1 promoter. Cells in slices from embryonic day 14 (E14), but not E13, mice displayed significant sex differences in their basal neuronal movement characteristics. Exposure to 10 nm estradiol-17beta (E2), but not 100 nm dihydrotestosterone, significantly altered cell movement characteristics within minutes of exposure, in a location-specific manner. E2 treatment decreased the rate of motion of cells located in the dorsal POA-AH but increased the frequency of movement in cells located more ventrally. These effects were consistent across age and sex. To further determine whether early-developing sex differences in the POA-AH depend upon gonadal steroids, we examined cell positions in mice with a disruption of the steroidogenic factor-1 gene, in which gonads do not form. An early-born cohort of cells were labelled with the mitotic indicator bromodeoxyuridine (BrdU) on E11. More cells were found in the POA-AH of females than males on the day of birth (P0) regardless of gonadal status. These results support the hypothesis that estrogen partially contributes to brain sexual dimorphism through its influence on cell movements during development. Estrogen's influence may be superimposed upon a pre-existing genetic bias.

Electrophoretic separation of large DNAs using steric confinement

We report an alternative method for electrophoretic separation of large DNAs using steric confinement between solid walls, without gel or obstacles. The change of electrophoretic mobility vs confinement thickness is investigated using fluorescence video microscopy. We observe separation at small confinement thicknesses followed by a transition to the bulk behavior (no separation) at a thickness of about 4 μm (a few radii of gyration for the studied DNA chains). We present tentative explanations of our original observations.

Early detection of nutrient and biotic stress in Phaseolus vulgaris

Prerequisites for optimal, high crop yield are disease‐free growth and an equilibrated supply of nutrients. Early signatures of stress‐altered physiology, before appearance of symptoms in the visible spectrum, allow timely treatment. Early detection of stress development was carried out on phaseolus vulgaris bean infected with the agriculturally important grey mould pathogen and under conditions of magnesium deficiency, limiting photosynthesis. During stress development, bean plants were monitored by time‐lapse imaging with thermal, video and chlorophyll fluorescence cameras, mounted on a gantry robot system. For early detection of grey mould infection, chlorophyll fluorescence imaging proved to be the most sensitive. This technique detected magnesium deficiency at least three days before visual symptoms appeared. Further development of non‐contact technology for plant health monitoring will help to achieve optimal productivity in greenhouse and field cultures. Associated establishment of a stress catalogue based on early symptoms will allow swift diagnosis.

LIM Kinase and Slingshot Are Critical for Neurite Extension

Cofilin and its closely related protein, actin-depolymerizing factor (ADF), are key regulators of actin cytoskeleton dynamics that have been implicated in growth cone motility and neurite extension. Cofilin/ADF are inactivated by LIM kinase (LIMK)-catalyzed phosphorylation and reactivated by Slingshot (SSH)-catalyzed dephosphorylation. Here we examined the roles of cofilin/ADF, LIMKs (LIMK1 and LIMK2), and SSHs (SSH1 and SSH2) in nerve growth factor (NGF)-induced neurite extension. Knockdown of cofilin/ADF by RNA interference almost completely inhibited NGF-induced neurite extension from PC12 cells, and double knockdown of SSH1/SSH2 significantly suppressed both NGF-induced cofilin/ADF dephosphorylation and neurite extension from PC12 cells, thus indicating that cofilin/ADF and their activating phosphatases SSH1/SSH2 are critical for neurite extension. Interestingly, NGF stimulated the activities of both LIMK1 and LIMK2 in PC12 cells, and suppression of LIMK1/LIMK2 expression or activity significantly reduced NGF-induced neurite extension from PC12 cells or chick dorsal root ganglion (DRG) neurons. Inhibition of LIMK1/LIMK2 activity reduced actin filament assembly in the peripheral region of the growth cone of chick DRG neurons. These results suggest that proper regulation of cofilin/ADF activities through control of phosphorylation by LIMKs and SSHs is critical for neurite extension and that LIMKs regulate actin filament assembly at the tip of the growth cone.

Protein transduction as a means of effective manipulation of Cdc42 activity in primary T cells

The Rho family GTPase Cdc42 is a critical regulator of cellular polarization from yeast to man. An analysis of its function in T cell activation is therefore of interest. This analysis poses two substantial challenges, similar to the analysis of many other critical T cell signaling intermediates. First, Cdc42 is required for development and cell survival, necessitating short-term manipulation of its activity. Second, Cdc42 is likely involved in multiple signaling pathways, requiring approaches to distinguish multiple roles. To address these challenges, we first determined and quantified spatio-temporal patterns of Cdc42 activity using live cell video fluorescence microscopy. This generates hypotheses at which times and locations Cdc42 might play possibly distinct roles. Second and as the focus of this manuscript, we employed protein transduction to manipulate Cdc42 activity for the generation of causality. Protein transduction allows such manipulation to be short-term, quantitative, and with multiple reagents. Here, we characterize uptake, retention, and subcellular distribution of protein transduction reagents. We describe how a more quantitative single cell analysis of Cdc42 activity provides superior distinction between experimental conditions. And we show how we have used dose responses of the protein transduction reagents to minimize side effects while retaining efficacy. We suggest that our strategy is an important complement to more established techniques to study protein function in primary T cells, in particular in the investigation of signaling intermediates that are essential for cell survival and regulate multiple aspects of T cell activation.

Micro-heterogeneity and micro-rheological properties of high-viscosity oat β-glucan solutions

Soluble fibre β-glucan is one of the key dietary materials in the healthy food products known for reducing serum cholesterol levels. The micro-structural heterogeneity and micro-rheology of high-viscosity oat β-glucan solutions were investigated by monitoring the thermally driven displacements of well-dispersed microspheres via video fluorescence microscopy. By comparing the distribution of the time-dependent mean-square displacement (MSD) and ensemble-averaged MSD of polystyrene microspheres imbedded in four concentrations of β-glucan solutions, we found that the solutions exhibited perfectly homogeneous behaviour at ⩽1%, but showed a certain degree of heterogeneity at 2%. Micro-rheology investigation revealed that β-glucan solutions displayed nearly perfect viscous behaviour at ⩽1%, but the property changed into viscoelastic at 2%. Both micro-structural heterogeneity and micro-rheological property shifts occurred over a small concentration range, between 1% and 2%, of β-glucan.

Real-Time Imaging of Platelet-Rich Thrombi in Thick-Walled Blood Vessels Using Near-Infrared Fluorescence Light.

Direct visualization of thrombus formation in real-time is limited by the fact that the vasculature is a closed system. Imaging of thrombi within arterioles and venules of small animals has been accomplished using fluorescent dyes that emit light in the visible spectrum. However, these agents are not able to penetrate large, thick-walled blood vessels and are therefore unable to detect thrombi in clinically important sites such as coronary, carotid, and femoral vessels. Near infrared light (NIR, 700–900 nm) demonstrates enhanced tissue penetration and less light scatter than visible light. By labeling platelets with IR-786, a heptamethine indocyanine-type fluorophore that emits NIR light, we have developed a reagent capable of detecting thrombi within thick-walled vessels of large animals. Optimal platelet loading occurred at 2 μM IR-786, resulting in an accumulation of 3 ×106 molecules/platelet. Normal platelet function was demonstrated by platelet aggregation and P-selectin surface expression studies. Clearance studies in 35 Kg Yorkshire pigs showed that IR-786-labeled platelets circulated for >2.5 h following infusion. IR-786-labeled platelets were next used in conjunction with an integrated NIR fluorescence video imaging system to digitally record thrombus formation in large vessels of pigs in real-time. Initially FeCl3 (oxidant injury) was utilized to induce clot formation in surgically exposed femoral arteries and IR-786-labeled platelets were shown to accumulate at the injury site with a signal to background ratio of 4.4±1.7. Thrombus growth was similarly detected and quantitated in coronary, carotid, and iliac arteries and veins. To assess potential applications of IR-786-labeled platelets for thrombus detection, thrombi were monitored in real-time, and quantified with respect to size and kinetics after electrocautery-induced injury to vessels, cutaneous incisions, intravascular stent insertion and introduction of embolic coils. Thrombolysis of formed IR-786-platelet-rich clots was also evaluated by the infusion of streptokinase and heparin, demonstrating dissolution of thrombi. Two color studies using IR-786-labeled platelets in conjunction with methylene blue, a near-infrared fluorescent blood pool agent used to assess vessel patency, showed that whilst the blood pool agent was more effective for assessing vessel patency, IR-786-labeled platelets were more sensitive for detecting and localizing thrombi within vessels. In addition, IR-786-labeled platelets were able to distinguish between actively growing and stabilized thrombi. IR-786-labeled platelets not only provide a useful tool to study mechanisms of thrombus formation but also afford a means of testing new anti-thrombotics and intravascular devices in vessels approximating the size of those in which clinically relevant thrombosis occurs. In addition, these pre-clinical studies indicate the utility of IR-786-labeled platelets as a contrast agent to detect intraoperative thrombosis, a complication of approximately 1–5% of vascular surgeries.

Nanokinetics of Drug Molecule Transport Into A Single Cell

To analyze drug transport at a single cell level, a mast cell line, RBL-2H3, was treated with cell-permeable fluorescent compounds, such as quinacrine, and was monitored by a fluorescence video microscope. Small areas in the video that corresponded to granules and part of the cytosol in a cell were chosen and the signal intensity in these areas was monitored sequentially. The initial rate of quinacrine uptake through the cell membrane calculated from the fluorescent signal was correlated with quinacrine concentration, and it decreased at a lower temperature, showing that the transport was an energy-requiring process, such as active transport. The kinetics of the transport through the microgranular membrane did not depend on the temperature but the pH in the cytosol, therefore this process should be passive transport by pH gradient. These data indicate that the observation of video microscope-mediated drug transport using fluorescent dye is useful in kinetic analysis at the nanometer scale.

Characterisation of rat corneal cells that take up soluble antigen: An in vivo and in vitro study

The aim of the present study was to determine the capacity of resident corneal and limbal dendritic cells (DC) and macrophages to capture antigen (Ag) in vivo and compare this to their capacity in vitro to take up Ag during organ culture conditions. To investigate Ag uptake in vivo 3 μl (30 μg) of fluorescently labelled Dextran, bovine serum albumin (BSA) or ovalbumin (OVA) were either placed on the intact ocular surface or injected into the anterior chamber (AC) or subconjunctival space of the Lewis rat eye. The presence of Ag + cells in the cornea was assessed using intravital fluorescence video microscopy. Animals were sacrificed 24 h after Ag injections or topical application and the distribution and phenotype of Ag + cells were analysed ex vivo by fluorescence and confocal microscopic analysis of immunostained and unstained corneal tissue wholemounts or frozen sections. Corneal buttons and corneoscleral rims from naive Lewis rats were placed in organ culture conditions in the presence of LPS with or without FITC-Dextran for 48 h and 72 h. The explants were examined by epi-fluorescence microscopy and the phenotype of Ag + cells in the supernatant from the organ cultures was analyzed by flow cytometry using a range of macrophage and DC markers. In vivo observations and microscopic examination of corneas 24 h following Ag topical application failed to reveal evidence of Ag retention by ocular cells. Those in which Ag had been placed in the AC or subconjunctival space revealed Ag + cells within the corneal stroma. The distribution of Ag + cells displayed a centripetal gradient, the most marked uptake of Ag being by cells in the circumferential limbal zone. Immunophenotypic studies revealed that Ag uptake was predominantly performed by cells that were CD68 +, CD172 + but rarely MHC class II +, a profile characteristic of macrophages. Occasional Ag + keratocytes were noted. In vitro studies of corneal buttons placed in culture conditions revealed that cells from the limbal zone, but not the central cornea, were able to take up Ag from the supernatant. Significant numbers of the cells that had migrated from the corneal buttons and captured fluorescent labelled Ag in the presence of LPS were revealed by flow cytometry to consist of CD163 + and CD11b + macrophages, but none expressed the DC markers CD11c or OX62 and they were also generally MHC class II −. In conclusion the present study revealed that macrophages and keratocytes in the corneal stroma and limbal episcleral tissue have the capacity to internalise fluorescent mock Ag injected into the AC or subconjunctival space or in culture conditions. The failure to demonstrate significant Ag trapping ability by corneal or limbal stromal or epithelial DC may either be due to the rarity of such cells or their lack of Ag trapping ability.

In vivo imaging of tumor growth after electrochemotherapy with cisplatin

Imaging methods can give both temporal and spatial dimensions to characterize the processes in progression of and/or treatment of specific disease Subcutaneous tumors can be cured after electrochemotherapy (ECT). Growth and reduction of tumors as a result of cytotoxic therapy can be followed by fluorescence video imaging directly on the same animal after treatment. Imaging of tumors should bring more information on the cellular effects of ECT. Green fluorescent protein (eGFP) expressing B16F10 and LPB tumors implanted in C57Bl/6 mice were treated with ECT with cisplatin. The growth or regression of the tumors was monitored either classically by using a caliper or by a manual definition of the region of interest where critical fluorescence levels were detected on the animals. A very good correlation between the two methods was observed. The eGFP mean fluorescence emission was only slightly affected by ECT with intravenously injected cisplatin. Ex vivo observations under a fluorescence microscope showed that eGFP was only detected on the outer layer of the tumor. No fluorescence was detected in the central part of the tumors, which were necrotic.

Dynamics of Individual Single-Walled Carbon Nanotubes in Water by Real-Time Visualization

Individual single-walled carbon nanotubes (SWNTs) in aqueous suspension are visualized directly by fluorescence video microscopy. The fluorescent tagging is simple, biocompatible, and does not modify the SWNTs. The dynamics of individual SWNTs in water are observed and quantified for the first time. We measure the confined rotational diffusion coefficient and find it in reasonable agreement with predictions based on confined diffusion of dilute Brownian rods. We determine the critical concentration at which SWNTs in suspensions start interacting. By analyzing the fluctuating shape of SWNTs in the 3 to 5 microm range, we determine that their persistence length ranges between 32 and 174 microm, in agreement with theoretical estimates; thus, commonly available SWNTs in liquids can be considered as rigid Brownian rods in the absence of imposed external fields or self-attractive forces.

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.

High-throughput design and fabrication of an integrated microsystem with high aspect-ratio sub-micron pillar arrays for free-solution micro capillary electrophoresis

A new technology approach for the design, fabrication and application of an integrated free-solution capillary electrophoresis microsystem is presented. Combining the advantages of projection, contact photolithography and deep-reactive-ion-etching, this approach allows fast and flexible formation of micron-sized channels integrated with extremely high aspect-ratio (>50:1) sub-micron pillar arrays on a silicon substrate. Utilizing fluorescence video microscopy, free-solution DNA separation has been demonstrated. Furthermore, the detailed DNA molecular interaction with the pillars inside the microsystem can be analysed. In comparison with the previously reported fabrication technologies, such as electron beam lithography, the newly presented technology approach offers a significant improvement in fabrication time and design flexibility; both are highly desirable not only for potential commercialization of the free-solution electrophoresis microsystem in applications such as lab-on-a-chip but also for systematic studies of micro-scale DNA kinetics.

Fluorescence imaging of sample zone narrowing and dispersion in a glass microchip: The effects of organic solvent (acetonitrile)–salt mixtures in the sample matrix and surfactant micelles in the running buffer

A mismatch in the EOF velocities between the sample zone and running buffer region is known to generate pressure-driven, parabolic flow profile of the sample plug in electrokinetic separation systems. In the present study, video fluorescence microscopy was employed to capture real-time dynamics of the sample plug (containing fluorescein as the probe molecule) in a discontinuous conductivity system within a glass microchip, in which the sample matrix consisted of a mixture of ACN and salt (NaCl), and the running buffer contained sodium cholate (SC) micelles as the pseudo-stationary phase (i.e., performing "ACN stacking" in the mode of MEKC). Upon application of the separation voltage, the video images revealed that zone narrowing and broadening of the probe molecules occurred as the sample plug headed toward the cathode during the initial time period, probably resulting in part from the stacking/sweeping, and destacking of the SC micelles at the boundaries between the sample zone and running buffer. Interestingly, a second sample zone narrowing event can be observed as the sample plug moved further toward the cathode, which could be attributed to the sweeping of the slower moving probe molecules by the faster moving SC micelles that originated from the anode. This phenomenon was studied as a function of pH, sample plug length, as well as the concentration of organic solvent and salt in the sample matrix. The data suggested that the presence of large amounts of an organic solvent (such as ACN or methanol) and salts in the sample matrix not only induces sample dispersion due to the formation of a pressure-driven (hydrodynamic) flow, but may also lead to the formation of a double sample zone narrowing phenomenon by altering the local EOF dynamics within the separation system.

Half-Sarcomere Dynamics in Myofibrils during Activation and Relaxation Studied by Tracking Fluorescent Markers

To study the dynamics of individual half-sarcomeres in striated muscle contraction, myofibrils prepared from rabbit psoas muscle and left ventricles of guinea pig were immunostained with two conjugated antibody complexes consisting of a primary antibody against either α-actinin or myomesin and a secondary fluorescently labeled Fab-fragment. We simultaneously measured force kinetics and determined the positions of the Z-line and M-band signals by fluorescence video microscopy and sophisticated computer vision (tracking) algorithms. Upon calcium activation, sarcomeres and half-sarcomeres shortened nonuniformly. Shortening occurred first rapidly and exponentially during the force rise and then slowly during the force plateau. In psoas myofibrils, time-resolved displacements of the A-band in sarcomeres were observed, i.e., the two halves of individual sarcomeres behaved nonuniformly. Nonuniformity in length changes between the two halves of sarcomeres was comparable to that between two adjacent half-sarcomeres of neighboring sarcomeres. Sequential lengthening of half-sarcomeres was observed in cardiac myofibrils during the rapid phase of force relaxation. The independent dynamics of the halves in a sarcomere reveals the half-sarcomere as the functional unit rather than the structural unit, the sarcomere. The technique will facilitate the study of filament sliding within individual half-sarcomeres and the mechanics of intersegmental chemomechanical coupling in multisegmental striated muscles.

Multiple‐Particle Tracking Study of Microheterogeneity of Nutrim‐10 Suspensions

ABSTRACTNutrim is a newly developed food product containing dietary soluble fiber β‐glucan. The microstructural heterogeneities of Nutrim‐10 suspensions were investigated by monitoring the thermally driven displacements of well‐dispersed microspheres through video fluorescence microscopy. By comparing the distribution of the time‐dependent mean‐square displacement (MSD) of polystyrene microspheres embedded in three concentrations of Nutrim‐10 suspensions, we found that the degree of heterogeneity of suspensions increased dramatically within a narrow range of Nutrim‐10 concentrations. The ensemble‐averaged MSD of 5.5% Nutrim‐10 suspension exhibited a power‐law behavior scaling linearly with time, which was similar to the behavior for a homogeneous aqueous glycerol solution. But the MSD distribution was wider and more asymmetric than for glycerol. Increasing Nutrim‐10 concentration rendered the MSD distribution much more asymmetric and skewed. This study provided a quantitative method to characterize the organization of Nutrim‐10 in suspension.

Influence of lipid shell physicochemical properties on ultrasound-induced microbubble destruction

We present the first study of the effects of monolayer shell physicochemical properties on the destruction of lipid-coated microbubbles during insonification with single, one-cycle pulses at 2.25 MHz and low-duty cycles. Shell cohesiveness was changed by varying phospholipid and emulsifier composition, and shell microstructure was controlled by postproduction processing. Individual microbubbles with initial resting diameters between 1 and 10 microm were isolated and recorded during pulsing with bright-field and fluorescence video microscopy. Microbubble destruction occurred through two modes: acoustic dissolution at 400 and 600 kPa and fragmentation at 800 kPa peak negative pressure. Lipid composition significantly impacted the acoustic dissolution rate, fragmentation propensity, and mechanism of excess lipid shedding. Less cohesive shells resulted in micron-scale or smaller particles of excess lipid material that shed either spontaneously or on the next pulse. Conversely, more cohesive shells resulted in the buildup of shell-associated lipid strands and globular aggregates of several microns in size; the latter showed a significant increase in total shell surface area and lability. Lipid-coated microbubbles were observed to reach a stable size over many pulses at intermediate acoustic pressures. Observations of shell microstructure between pulses allowed interpretation of the state of the shell during oscillation. We briefly discuss the implications of these results for therapeutic and diagnostic applications involving lipid-coated microbubbles as ultrasound contrast agents and drug/gene delivery vehicles.

Self-diffusion of particles in gas-driven granular layers with periodic flow modulation

We study self-diffusion of particles in gas-driven granular layers by high-speed fluorescent video microscopy. We show that periodic flow modulation results in an enhancement of the particle's diffusion. The diffusion enhancement, which in turn is an indication of more efficient fluidization of the granular layer, is associated with the onset of disordered subharmonic patterns. Our measurements provide a sensitive characterization method of the fluidization properties of particulate-gas systems.

Somatostatin inhibits insulin secretion from pancreatic B-cells through modulation of R-type Ca2+ channel activity

Background: Somatostatin (SST) inhibits insulin secretion from pancreatic B-cells by modulating the activity of Ca2+ permeable channels. Five pharmacologically distinctive subtypes of voltage-operated calcium channels (VOOCs) are known: L-type [CaV1.1–1.4], P/Q-type [CaV2.1], N-type [CaV2.2; R-type [CaV2.3] and T-type [CaV3.1–3.3]). Recently it has been demonstrated that the R-type VOCC subtype plays an important role at regulating the second phase of insulin secretion. Its involvement in SST-dependent regulation of insulin secretion is not known. Aims: To evaluate whether the R-type channel confers the effects of SST and somatostatin receptor subtype (SSTR)-selective agonists on insulin secretion from pancreatic B-cells. Materials & Methods: The effects of SST were determined in fura2-loaded INS-1 cells, a permanent model of insulin-secreting B-cells, using a fluorescence video imaging system. INS-1 were incubated with SST with or without the specific R-type channel blocker SNX-482 (10 nM) and the levels of intracellular Ca2+ concentration ([Ca2+]i) were determined. The effects of SST and SSTR-selective agonists on insulin secretion, in the presence or absence of SNX-482 were measured by an ELISA. Results: Ca2+ responses in INS-1 cells were clearly detectable at high glucose levels (10 and 20 mM) as compared to 5 mM glucose. SST (100 nM) transiently increased [Ca2+]i within 5min, which declined after 15–30min below the baseline (e.g. 15min: 95±2% of control (set to 100%) (n=8; p<0.05)). Combination of SST with the R-type channel blocker SNX-482 resulted in a smaller transient [Ca2+]i increase followed by a much faster decrease below the baseline. The inhibitory effect of SST and SSTR-selective agonists on 20 mM glucose-induced insulin secretion was strongly reduced in the presence of SNX-482. Conclusion: These data demonstrate that “neuronal“ R-type channel (CaV2.3) plays an important role at mediating the inhibitory effects of somatostatin on insulin secretion.

Platelet Adhesion Via Glycoprotein IIb Integrin Is Critical for Atheroprogression and Focal Cerebral Ischemia

The platelet glycoprotein (GP) IIb/IIIa integrin binds to fibrinogen and thereby mediates platelet aggregation. Here, we addressed the role of GP IIb for platelet adhesion and determined the relevance of platelet GP IIb for the processes of atherosclerosis and cerebral ischemia-reperfusion (I/R) injury. GP IIb(-/-) mice were generated and bred with ApoE(-/-) animals to create GP IIb(-/-)ApoE(-/-) mice. Platelet adhesion to the mechanically injured or atherosclerotic vessel wall was monitored by in vivo video fluorescence microscopy. In the presence of GP IIb, vascular injury and early atherosclerosis induced platelet adhesion in the carotid artery (CA). In contrast, platelet adhesion was significantly reduced in the absence of GP IIb integrin (P<0.05). To address the contribution of platelet GP IIb to atheroprogression, we determined atherosclerotic lesion formation in the CA and aortic arch (AA) of GP IIb(+/+)ApoE(-/-) or GP IIb(-/-)ApoE(-/-) mice. Interestingly, the absence of GP IIb attenuated lesion formation in CA and AA, indicating that platelets, via GP IIb, contribute substantially to atherosclerosis. Next, we assessed the implication of GP IIb for cerebral I/R injury. We observed that after occlusion of the middle cerebral artery, the cerebral infarct size was drastically reduced in mice lacking GP IIb compared with wild-types. These findings show for the first time in vivo that GP IIb not only mediates platelet aggregation but also triggers platelet adhesion to exposed extracellular matrices and dysfunctional endothelial cells. In a process strictly involving GP IIb, platelets, which are among the first blood cells to arrive at the scene of endothelial dysfunction, contribute essentially to atherosclerosis and cerebral I/R injury.