Disorganization of the cytoskeleton of neurons has major consequences on the transport of neurotransmitters via the microtubule network. The interaction of cytoskeleton proteins (actin and tubulin) was studied in neuronal SK-N-BE cells treated with tetracosanoic acid (C24:0), which is cytotoxic and increased in Alzheimer's disease patients. When SK-N-BE cells were treated with C24:0, mitochondrial dysfunctions and a non-apoptotic mode of cell death were observed. Fluorescence microscopy revealed shrunken cells with perinuclear condensation of actin and tubulin. Impact of C24:0 on actin-microtubule interaction in human neuronal SK-N-BE cells: evaluation by FRET confocal spectral imaging microscopy after dual staining with rhodamine-phalloidin and tubulin tracker green After staining with rhodamine-phalloidin and with an antibody raised against α-/β-tubulin, modifications of F-actin and α-/β-tubulin levels were detected by flow cytometry. Lower levels of α-tubulin were found by Western blotting. In C24:0-treated cells, spectral analysis and fluorescence recovery after photobleaching (FRAP) measured by confocal microscopy proved the existence of fluorescence resonance energy transfer (FRET) when actin and tubulin were stained with tubulin tracker and rhodamine-phalloidin demonstrating actin and tubulin co-localization/interaction. In control cells, no FRET was observed. Our data demonstrate quantitative changes in actin and tubulin, and modified interactions between actin and tubulin in SK-N-BE cells treated with C24:0. They also show that FRET confocal imaging microscopy is an interesting method for specifying the impact of cytotoxic compounds on cytoskeleton proteins.

In the present study, we make use of the ability of two-photon confocal laser scanning microscopes (CLSMs) equipped with tunable lasers to produce spectral excitation image sequences. Furthermore, unmixing, which is usually performed on emission image sequences, is performed on these excitation image sequences. We use factor analysis of medical image sequences (FAMIS), which produces factor images, to unmix spectral image sequences of stained structures in tissue sections to provide images of characterized stained cellular structures. This new approach is applied to histological tissue sections of mouse aorta containing labeled iron nanoparticles stained with Texas Red and counterstained with SYTO13, to obtain visual information about the accumulation of these nanoparticles in the arterial wall. The possible presence of Texas Red is determined using a two-photon CLSM associated with FAMIS via the excitation spectra. Texas Red and SYTO13 are thus differentiated, and corresponding factor images specify their possible presence and cellular localization. In conclusion, the designed protocol shows that sequences of images obtained by excitation in a two-photon CLSM enables characterization of Texas Red-stained nanoparticles and other markers. This methodology offers an alternative and complementary solution to the conventional use of emission spectra unmixing to localize fluorescent nanoparticles in tissue samples.

In the context of multiple sclerosis and X-linked adrenoleukodystrophy, 7-ketocholesterol (7KC) and very long chain fatty acids (C24:0, C26:0) are supposed to induce side effects respectively on oligodendrocytes which are myelin (which is a lipoproteic complex) synthesizing cells. The effects of 7KC (25, 50 μM), C24:0 and C26:0 (10, 20 μM) on cell viability and lipid membrane organization were investigated on 158N murine oligodendrocytes. Concerning 7KC and fatty acids (at 20 μM only): 1) cell growth was strongly inhibited; 2) marked induction of cell death was revealed with propidium iodide (PI); 3) no apoptotic cells were found with C24:0 and C26:0 (absence of cells with condensed and/or fragmented nuclei, of FLICA positive cells and of PI negative/SYTO16 negative cells); 4) some apoptotic cells were detected with 7KC. Fatty acids (at 20 μM only) and 7KC also induced a disorganization of lipid membranes revealed with Merocyanine 540. So, to point out the effects of 7KC (25 μM), C24:0 and C26:0 (20 μM) on the lateral organization of lipid membranes, we used LAURDAN, which gives simultaneous information about morphology and phase state of lipid domains: its emission is blue in the ordered lipid phase, green in the disordered lipid phase. To overcome the qualitative filtering settings of blue and green emission colors, data obtained by mono- and bi-photon confocal microscopy were analyzed by spectral analysis. Sequences of emission images were obtained on both mono- and bi-photon confocal microscopes and processed by means of Factor Analysis of Medical Image Sequences (FAMIS), which is a relevant tool to unmix emission spectra and provide pure color images. Only 7KC was capable to induce a green emission with LAURDAN. Thus, at concentrations inducing oligodendrocyte cell death, 7KC (25 μM) is more efficient than C24:0 and C26:0 (20 μM), to trigger lateral lipid membrane disorganization.

OBJECTIVE:To evaluate the cytotoxicity of iron nanoparticles on cardiac cells and to determine whether they can modulate the biological activity of 7-ketocholesterol (7KC) involved in the development of cardiovascular diseases. Nanoparticles of iron labeled with Texas Red are introduced in cultures of nonbeating mouse cardiac cells (HL1-NB) with or without 7-ketocholesterol 7KC, and their ability to induce cell death, pro-inflammatory and oxidative effects are analyzed simultaneously.STUDY DESIGN:Flow cytometry (FCM), confocal laser scanning microscopy (CLSM), and subsequent factor analysis image processing (FAMIS) are used to characterize the action of iron nanoparticles and to define their cytotoxicity which is evaluated by enhanced permeability to SYTOX Green, and release of lactate deshydrogenase (LDH). Pro-inflammatory effects are estimated by ELISA in order to quantify IL-8 and MCP-1 secretions. Pro-oxidative effects are measured with hydroethydine (HE).RESULTS:Iron Texas Red nanoparticles accumulate at the cytoplasmic membrane level. They induce a slight LDH release, and have no inflammatory or oxidative effects. However, they enhance the cytotoxic, pro-inflammatory and oxidative effects of 7KC. The accumulation dynamics of SYTOX Green in cells is measured by CLSM to characterize the toxicity of nanoparticles. The emission spectra of SYTOX Green and nanoparticles are differentiated, and corresponding factor images specify the possible capture and cellular localization of nanoparticles in cells.CONCLUSION:The designed protocol makes it possible to show how Iron Texas Red nanoparticles are captured by cardiomyocytes. Interestingly, whereas these fluorescent iron nanoparticles have no cytotoxic, pro-inflammatory or oxidative activities, they enhance the side effects of 7KC.

OBJECTIVE:To evaluate the capture of nanoparticles (quantum dots [QDs], fluorospheres) by nonbeating mouse cardiac cells (HL1-NB) cultured without or with 7-ketocholesterol (7KC) found at an increased level in the plasma of atherosclerotic patients and to simultaneously analyze their cytotoxic, proinflammatory and oxidative properties. STUDY DESIGN:Flow cytometry (FCM), confocal laser scanning microscopy and subsequent factor analysis image processing were used to characterize the uptake of nanoparticles and to define their cytotoxicity, evaluated by enhanced permeability to SYTOX Green, release of lactate dehydrogenase (LDH) and morphologic nuclear changes determined with Hoechst 33342. Proinflammatory effects were estimated by enzyme linked immunoassay to quantify IL-8 and MCP-1 secretion. The overproduction of reactive oxygen species (ROS) was determined by FCM with hydroethidine. RESULTS:Whereas the nanoparticles had no cytotoxic or inflammatory effects, they could stimulate ROS production. QDs were not incorporated. When 7KC was used, LDH release was enhanced and QDs potentialized IL-8 secretion. The incorporation and exit dynamics of nanoparticles were visualized to differentiate the emission spectra of SYTOX Green and nanoparticles and to precisely determine the cellular localization of nanoparticles. CONCLUSION:The selected nanoparticles, which accumulate at the inner or outer cytoplasmic membrane level, can induce biologic activities and are able to interfere with those of chemically defined molecules such as 7KC.

Among the oxysterols accumulating in atherosclerotic plaque, 7-ketocholesterol (7KC) is a potent apoptotic inducer, which favours myelin figure formation and polar lipid accumulation. This investigation performed on U937 cells consisted in characterizing the myelin figure formation process; determining the effects of 7KC on the PI3-K/PDK-1/Akt signalling pathway; evaluating the activities of vitamin E (Vit-E) (α-tocopherol) on the formation of myelin figures and the PI3-K/PDK-1/Akt signalling pathway and assessing the effects of PI3-K inhibitors (LY-294002, 3-methyladenine) on the activity of Vit-E on cell death and polar lipid accumulation. The ultrastructural and biochemical characteristics of myelin figures (multilamellar cytoplasmic inclusions rich in phospholipids and 7KC present in acidic vesicles and the reversibility of these alterations) support the hypothesis that 7KC is an inducer of phospholipidosis. This oxysterol also induces important changes in lipid content and/or organization of the cytoplasmic membrane demonstrated with merocyanine 540 and fluorescence anisotropy, a loss of PI3-K activity and dephosphorylation of PDK-1 and Akt. It is noteworthy that Vit-E was able to counteract phospholipidosis and certain apoptotic associated events (caspase activation, lysosomal degradation) to restore PI3-K activity and to prevent PDK-1 and Akt dephosphorylation. When Vit-E was associated with LY-294002 or 3-methyladenine, impairment of 7KC-induced apoptosis was inhibited, and accumulation of polar lipids was less counteracted. Thus, 7KC-induced apoptosis is a PI3-K-dependent event, and Vit-E up- and down-regulates PI3-K activity and phospholipidosis, respectively.

Resveratrol is a well known polyphenol largely produced in grapevine. It is a strong antioxidant and a free radical scavenger. It exhibits several beneficial effects for health including cancer. Resveratrol antioxidant activity is essential in the prevention of chemical-induced cancer by inhibiting initiation step of carcinogenesis process but it is also considered to inhibit cancer promotion and progression steps.While the effects of resveratrol on cancer cells are widely described, the data available on the antiproliferative potential of resveratrol derivatives remain weak. Nevertheless, resveratrol analogs could exhibit stronger potentials than the parent molecule. So, we compared the cellular effects of trans-resveratrol, trans-epsilon-viniferin and their respective acetate derivatives, as well as a polyphenol mixture extracted from grapevine shoots, called vineatrol. We studied their abilities to interfere with cell proliferation, their uptake and their effects on parameters of cellular state in human hepatoma cells (HepG2).Cell growth experiments show that resveratrol triacetate presents a slightly better antiproliferative potential than resveratrol. The dimer epsilon-viniferin,as well as its pentaacetate analog, is less powerful than resveratrol, although a similar uptake kinetics in cells. Interestingly, among the tested polyphenols, vineatrol is the most potent solution, indicating a possible synergistic effect of both resveratrol and epsilon-viniferin. We took advantage of the fluorescence properties of these compounds to evidence cellular uptake by using flow cytometry. In addition, by competition assay, we demonstrate that resveratrol triacetate enters in hepatic HepG2 cells by the same way as resveratrol. By autofluorescence in situ measurement we observed that resveratrol and related compounds induce deep changes in cells activity. These changes occur mainly by increasing NADPH cell content and the number of green fluorescent cytoplasmic granular structures which may be related to an induction of detoxifying enzyme mechanisms. (c) 2008 Elsevier Masson SAS. All rights reserved.

The 7-ketocholesterol (7KC)-induced cell death has some characteristics of apoptosis and is associated with polar lipid accumulation. So, we investigated the effects of the broad-spectrum caspase inhibitor z-VAD-fmk and of the caspase-2 inhibitor z-VDVAD-fmk on lipid profile evaluated by staining with Nile Red (NR). The 7KC-treated human monocytic U937 cells were cultured in the absence or in the presence of the 2 caspase inhibitors z-VAD-fmk or z-VDVAD-fmk. When staining with NR is performed, neutral and polar lipids have yellow and orange/red emission, respectively, and fluorescence was then analyzed by flow cytometry (FCM) and by confocal laser scanning microscopy (CLSM) combined with subsequent image processing. The 3D-image sequences were obtained by means of CLSM using spectral analysis, and were analyzed by the factor analysis of medical image sequences algorithm to differentiate spectra inside mixed fluorescence emission and get corresponding specific images. By FCM, comparatively to untreated cells, higher percentages of red fluorescent cells were identified in 7KC-treated cells. Factor curves and images reveal orange and red fluorescence emissions in 7KC-treated cells and show yellow, orange, and red fluorescence emissions in 7KC-treated cells cultured in the presence of z-VAD-fmk or z-VDVAD-fmk. Our data support that investigation by FCM and by spectral analysis in CLSM associated with subsequent image processing provides useful tools to determine the effect of caspase inhibitors on lipid content evaluated with NR. They also favor the hypothesis of relationships between caspase activity and polar lipid accumulation. (C) 2007 International Society for Analytical Cytology

L’article développe une méthodologie de recherche basée sur l’utilisation de marqueurs fluorescents applicable aux préparations cellulaires ou tissulaires marquées, pour l’observation sur des microscopes confocaux à balayage laser.

Oxysterols, mainly those oxidized at the C7 position, induce a complex mode of cell death exhibiting some characteristics of apoptosis associated with a rapid induction of lipid rich multilamellar cytoplasmic structures (myelin figures) observed in various pathologies including atherosclerosis. The aim of this study was to determine the relationships between myelin figure formation, cell death, and lipid accumulation in various cell lines (U937, THP-1, MCF-7 [caspase-3 deficient], A7R5) treated either with oxysterols (7-ketocholesterol [7KC], 7β-hydroxycholesterol, cholesterol-5α,6α-epoxide, cholesterol-5β,6β-epoxide, 25-hydroxycholesterol) or cytotoxic drugs (etoposide, daunorubicin, tunicamycin, rapamycin). Cell death was assessed by the measurement of cellular permeability with propidium iodide, characterization of the morphological aspect of the nuclei with Hoechst 33342, and identification of myelin figures by transmission electron microscopy. Nile Red staining (distinguishing neutral and polar lipids) was used to identify lipid content by flow cytometry and spectral imaging microscopy. Whatever the cells considered, myelin figures were only observed with cytotoxic oxysterols (7KC, 7β-hydroxycholesterol, cholesterol-5β, 6β-epoxide), and their formation was not inhibited by the broad spectrum caspase inhibitor z-VAD-fmk. When U937 cells were treated with oxysterols or cytotoxic drugs, polar lipid accumulation was mainly observed with 7KC and 7β-hydroxycholesterol. The highest polar lipid accumulation, which was triggered by 7KC, was counteracted by z-VAD-fmk. These findings demonstrate that myelin figure formation is a caspase-independent event closely linked with the cytotoxicity of oxysterols, and they highlight a relationship between caspase activity and polar lipid accumulation.

OBJECTIVE: To evaluate CD36 expression with quantum dots 605 (QDs 605) on untreated and 7-ketocholesterol (7KC)-treated monocytic U937 cells by flow cytometry (FCM) and confocal and multiphoton laser scanning microscopy (CLSM).STUDY DESIGN. Cells were analyzed by CLSM, following flow cytometric quantification of CD36 expression and 7KC uptake. Image sequences were obtained by spectral analysis in monophoton and multiphoton CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm to differentiate emission spectra. In CLSM analysis, cell deposits were screened in ultraviolet excitation modes to optimize the possibilities of QDs 605 and have the benefit of nuclei counterstaining by DAPI.RESULTS: FCM and CLSM reveal the expression of CD36 by means of QDs 605. FCM provides information on 7KC uptake. CLSM provides the localization of 7KC vs. DAPI. As factor curves and images show the red, narrow emission of QDs 605 vs. violet and blue emissions of 7KC and DAPI, respectively, a reliable identification of CD36 is obtained.CONCLUSION: QDs 605 are useful tools to perform antigenic expression in FCM and CLSM. Moreover, CLSM and subsequent spectral analysis provide a more specific characterization of QDs 605 fluorescent emission in the UV excitation mode and a simultaneous identification of 7KC.

Here, we identify fetal bone marrow (BM)-derived CD34hiCD45RAhiCD7+ hematopoietic progenitors as thymus-colonizing cells. This population, virtually absent from the fetal liver (FL), emerges in the BM by development weeks 8-9, where it accumulates throughout the second trimester, to finally decline around birth. Based on phenotypic, molecular, and functional criteria, we demonstrate that CD34hiCD45RAhiCD7+ cells represent the direct precursors of the most immature CD34hiCD1a- fetal thymocytes that follow a similar dynamics pattern during fetal and early postnatal development. Histological analysis of fetal thymuses further reveals that early immigrants predominantly localize in the perivascular areas of the cortex, where they form a lymphostromal complex with thymic epithelial cells (TECs) driving their rapid specification toward the T lineage. Finally, using an ex vivo xenogeneic thymus-colonization assay, we show that BM-derived CD34hiCD45RAhiCD7+ progenitors are selectively recruited into the thymus parenchyma in the absence of exogenous cytokines, where they adopt a definitive T cell fate.

OBJECTIVE:To demonstrate CD36 expression with quantum dots (QDs) 525 and/or 605 on human monocytic U937 cells and atherosclerotic tissue sections by means of flow cytometry (FCM) and/or confocal laser scanning microscopy (CLSM).STUDY DESIGN:U937 cells and tissue sections were analyzed by means of FCM and/or CLSM. FCM was performed, using different ultraviolet (UV) and visible (488/532 nm) excitation modes. In the visible mode, fluorescence intensities of QDs, phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were compared. Three-dimensional (3-D) sequences of images were obtained by spectral analysis in a CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm, providing factor curves and images. Factor images are the result of the FAMIS image processing method, which differentiates emission spectra from 3D sequences of images. In CLSM analysis, preparations are screened in a UV excitation mode to optimize the possibilities of QDs and have the benefit of 4',6-diamino-2-phenylindole or Hoechst 33342 counterstaining of nuclei.RESULTS:FCM and CLSM revealed CD36 expression by means of QDs 525 and/or 605. Fluorescence intensity of PE and of FITC was higher than that of QDs 525 and of 605. As factor curves and images show the red emission of QDs 605 only, subsequent reliable identification and localization of CD36 was obtained.CONCLUSION:QDs 525 and 605 are useful to analyze antigenic expression. Following FCM, which is well adapted to detect fluorescence emission of QDs in the UV or visible excitation mode, CLSM and subsequent spectral analysis assess more specific characterization of QD fluorescent emissions.

Contrast-enhanced (CE) MRI provides in vivo physiological information that cannot be obtained by conventional imaging methods. This information is generally extracted by using models to represent the circulation of contrast agent in the body. However, the results depend on the quality of the fit obtained with the chosen model. Therefore, one must check the fit quality to avoid working on physiologically irrelevant parameters. In this study two dimensionless criteria-the fraction of modeling information (FMI) and the fraction of residual information (FRI)-are proposed to identify errors caused by poor fit. These are compared with more conventional criteria, namely the quadratic error and the correlation coefficient, both theoretically and with the use of simulated and real CE-MRI data. The results indicate the superiority of the new criteria. It is also shown that these new criteria can be used to detect oversimplified models.

Background: Oxidized low-density lipoproteins play key roles in atherosclerosis. Their toxicity is at least in part due to 7-ketocholesterol (7KC), which is a potent inducer of apoptosis. In this study on human promonocytic U937 cells, we determined the effects and the interactions of 7KC with cellular lipids during 7KC-induced apoptosis.Methods: Morphologic and functional changes were investigated by microscopic and flow cytometric methods after staining with propidium iodide, 3,3'-dihexyloxacarbocyanine iodide, and Hoechst 33342. Cellular lipid content was identified by using filipin to quantify free cholesterol and Nile Red (NR), which emit a yellow or orange-red fluorescence in the presence of neutral and polar lipids, respectively. After staining with NR, interactions of 7KC with cellular lipids were identified by fluorescence resonance energy transfer biphoton spectral imaging confocal microscopy and by subcellular fractionation, gas chromatography, and mass spectrometry.Results: During 7KC-induced apoptosis the fluorescence from filipin and the ratio of measured (orange-red vs. yellow) fluorescence of NR were enhanced. Spectral analysis of images obtained in biphoton mode and resulting factor images demonstrated the occurrence of fluorescence resonance energy transfer between 7KC and NR and the subsequent colocalization of 7KC and NR. These data were in agreement with biochemical characterization and demonstrated that 7KC and neutral and polar lipids accumulate in NR-stained cytoplasmic structures.Conclusions: During 7KC-induced apoptosis, 7KC modifies the cellular content of neutral and polar lipids, favors free cholesterol accumulation, and colocalizes with neutral and polar lipids that are inside NR-stained cytoplasmic structures. (c) 2005 Wiley-Liss, Inc.

PURPOSE:To evaluate a magnetic resonance (MR) imaging contrast agent for tumor detection based on paramagnetic nonionic vesicles (niosomes) bearing polyethylene glycol (PEG) and glucose conjugates for the targeting of overexpressed glucose receptors.MATERIALS AND METHODS:Four gadobenate dimeglumine-loaded niosome preparations including nonconjugated niosomes, niosomes bearing glucose conjugates (N-palmitoyl glucosamine [NPG]), niosomes bearing PEG 4400, and niosomes bearing both PEG and NPG were tested. In vitro cellular uptake was measured at electron paramagnetic resonance (EPR) after incubation with human prostate carcinoma, PC3, cells. In vivo distribution was studied at MR imaging 6, 12, and 24 hours after injection, with assessment of tumor, brain, liver, and muscle signal intensity (SI) in 49 mice bearing PC3 cells. Efficiency of targeted contrast agents was assessed with tumor-to-muscle contrast-to-noise ratio (CNR). Testing for differences was performed with analysis of variance followed by a posteriori Fisher test.RESULTS:In vitro, gadolinium could be detected at EPR only in cell pellets incubated with niosomes bearing glucose conjugates or niosomes bearing both glucose conjugates and PEG (4.9. 10(-15) and 4.5. 10(-15) mol gadolinium per PC3 cell). In vivo, marked predominant tumor enhancement was demonstrated 24 hours after injection of glycosylated PEG niosomes (P <.01); no significant differences were observed following injection of nonconjugated niosomes, glycosylated niosomes, or PEG 4400 niosomes. Twenty-four hours after injection, sole presence of NPG or PEG 4400 on the surface of the niosome led to higher tumor-to-muscle CNR than that observed after injection of nonconjugated niosomes (CNR of 3.3 +/- 0.7 [SD], 3.4 +/- 2.2, and 0 +/- 1.9). Combination of NPG and PEG led to even higher tumor-to-muscle CNR (6.3 +/- 2.2).CONCLUSION:Combination of PEG and glucose conjugates on the surface of niosomes significantly improved tumor targeting of an encapsulated paramagnetic agent assessed with MR imaging in a human carcinoma xenograft model.

OBJECTIVE:To demonstrate that cellular preparations requiring color analysis of different domains stained by molecular cytogenetic methods (fluorescence in situ hybridization) can be processed by spectral analysis of fluorescent emissions by either factor analysis of medical image sequences (FAMIS) or a META confocal configuration to isolate fluorescent probes.STUDY DESIGN:Three-dimensional sequences of images obtained by spectral analysis in a META confocal microscope (Carl Zeiss SAS, Jena, Germany) were analyzed by META processing and the FAMIS algorithm, which provides factor curves. META and factor images were then the result of image-processing methods that cover emission spectra.RESULTS:Factor curves and factor or META images can help to analyze targets inside nuclei.CONCLUSION:It is possible to process preparations containing numerous spots on different colors to differentiate stained targets and to improve visualization and detection.