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International audience; We explored the mechanisms underlying microglia cell-carbon nanotube interactions in order to investigate whether electrical properties of Carbon-Nanotubes (CNTs) could affect microglia brain cells function and phenotype. We analyzed the effects induced by highly electro-conductive Multi-Walled-Carbon-Nanotubes (a-MWCNTs), on microglia cells from rat brain cortex and compared the results with those obtained with as prepared not conductive MWCNTs (MWCNTs) and redox-active Double-Walled-Carbon-Nanotubes (DWCNTs). Cell viability and CNT capacity to stimulate the release of nitric oxide (NO), pro-inflammatory (IL-1b, TNF-a) and anti-inflammatory (IL-10, TGF-b1) cytokines and neurotrophic factors (mNGF) were assessed. Electro-conductive MWCNTs, besides not being cytotoxic, were shown to stimulate, at 24 h cell exposure, classical "M100 microglia activation phenotype, increasing significantly the release of the main pro-inflammatory cytokines. Conversely, after 48 h cell exposure, they induced the transition from classical "M100 to alternative "M200 microglia phenotype, supported by anti-inflammatory cytokines and neuroprotective factor mNGF release. The analysis of cell morphology change, by tubulin and CD-206 þ labelling showed that M2 phenotype was much more expressed at 48 h in cells exposed to a-MWCNTs than in untreated cells. Our data suggest that the intrinsic electrical properties of CNTs could be exploited to modulate microglia phenotype and function stimulating microglia anti-inflammatory potential.

Amyloid beta protein (Abeta) fibrillogenesis is considered one of the crucial steps of Alzheimer's disease (AD) pathogenesis. The effect of endogenous neuronal amyloid fibrils on memory processes is unknown. To investigate this issue, we first characterised the Abeta fibrillar aggregates secreted by cerebellar granule cells and then we evaluated the effect of neuronal fibrils on an invertebrate model of memory. An increase of fibril formation, assessed by Thioflavin T (ThT) fluorescence, was observed in the conditioned medium of apoptotic neurons during 48 h of the apoptotic process. Moreover, the depolarisation-stimulated secretion of cerebellar granule cells contains monomers of endogenous Abeta, which undergo cell-free fibrillogenesis over several days of incubation. The pattern of single endogenous fibrils, examined by electron microscopy, was similar to that of synthetic Abeta while a tighter and more complex interfibrillar organization was observed in endogenous fibrils. The biological effect of neuronal fibrils was studied in a long-term memory (LTM) paradigm, namely the context-signal learning of the crab Chasmagnathus. Pre-training injection of neuronal fibril extract (protein concentration, 1 microg/ml) induced amnesia in a dose-dependent manner. On the contrary, no effect on retention was observed with the administration of two orders higher doses (100 microg/ml) of synthetic Abeta1-40. These results indicate that only naturally secreted fibrils, but not synthetic Abeta, clearly interfere with memory process.

The fluorescent membrane probe 6-propionyl-2-dimethylaminonaphthalene (Prodan) displays a high sensitivity to the polarity and packing properties of lipid membrane. Contrary to 6-lauroyl-2-dimethylaminonaphthalene (Laurdan), Prodan can also monitor the properties of the membrane surface, i.e., the polar-head pretransition. In bilayers composed of coexisting gel and liquid-crystalline phases, Prodan shows a preferential partitioning in the latter, so that the detected membrane properties mainly belong to fluid domains. In the presence of cholesterol, the packing properties of the gel phase phospholipids are modified in such a way that Prodan can penetrate and label the membrane. Although Prodan labeling of the gel phase is a function of cholesterol concentration, 3 mol percent cholesterol is sufficient for a 60% Prodan labeling with respect to the maximum labeling reached at 15 mol percent cholesterol. We present steady-state and dynamical fluorescence measurements of Prodan in bilayers in the presence of cholesterol. Our results fit the liquid-ordered/liquid-disordered phase model for cholesterol-containing membranes and show that the presence of cholesterol, in addition to modification to the phase state of the hydrophobic portion of the bilayer, strongly affects the packing and the polarity of the membrane hydrophobic-hydrophilic interface. © 2001 Elsevier Science B.V.

Among different proposed mechanisms to account for the protection exerted by estrogens against cardiovascular diseases, the antioxidant effect has attracted considerable attention. We confirmed that 17-beta-estradiol (E2), when added to human LDL at a 6:1 ratio to apoB-100, markedly delays the phase of massive LDL lipid peroxidation induced by Cu(2+). We also observed an increased oxidative resistance of E2-treated LDL by monitoring the early phase of oxidative degradation on the basis of increased LDL surface polarity by the generalized polarization of the lipophilic fluorescent probe 2-(dimethylamino)-6-lauroylnaphthalene (Laurdan). A scavenging of free radicals by E2 is ruled out since, consistent with its structure, its rate constant for the reduction of peroxy radicals is extremely low, i.e., 0.02% of that of vitamin E. Tryptophan fluorescence lifetime and circular dichroism measurements revealed that (i) apoB-100 undergoes a conformational modification and a progressive loss of secondary structure during lipid peroxidation; (ii) E2 increases apoB-100 secondary structure and modifies its conformation; and (iii) the apoB-100 conformational change induced by E2 makes this protein resistant to modifications brought about by lipid peroxidation. We propose that E2, by affecting apoB-100 secondary structure and conformation, modifies the interaction of this protein with the outer layer of the LDL particle thus increasing its overall oxidative resistance.

Fluorescence spectral features of 6-propionyl-2-dimethylaminonaphthalene (Prodan) in phospholipid vesicles of different phase states are investigated. Like the spectra of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan), the steady-state excitation and emission spectra of Prodan are sensitive to the polarity of the environment, showing a relevant shift due to the dipolar relaxation phenomenon. Because of the different lengths of their acyl residues, the partitioning of the two probes between water and the membrane bilayer differs profoundly. To account for the contribution of Prodan fluorescence arising from water, we introduce a three-wavelength generalized polarization method that makes it possible to separate the spectral properties of Prodan in the lipid phase and in water, and to determine the probe partitioning between phospholipid and water and between the gel and the liquid-crystalline phases of phospholipids. In contrast to Laurdan, Prodan preferentially partitions in the liquid-crystalline phase with respect to the gel and is sensitive to the polar head pretransition, and its partition coefficient between the membrane and water depends on the phase state, i.e., on the packing of the bilayer. Prodan is sensitive to polarity variations occurring closer to the bilayer surface than those detected by Laurdan.

Established microscopies such as Scanning Electron Microscopy (SEM) and more recent developments such as Atomic Force Microscopy (AFM) and X-ray Photo-Electron Emission spectroMicroscopy (X-PEEM) can only image the sample surface. We present an argon sputtering method able to progressively expose inner cell structures without apparent damage. By varying the sputtering time, the structure of cell cytoskeleton, vesicles, mitochondria, nuclear membrane, and nucleoli can be imaged. We compared images obtained with confocal fluorescence microscopy, transmission electron microscopy (TEM), SEM, and X-PEEM on similar samples after argon sputtering, then confirmed the similarity of reference intracellular structures, including cytoskeleton fibers, cell-cell and cell-substrate adhesion structures, and secretory vesicles. We conclude that the sputtering method is a new valuable tool for surface sensitive microscopies.

Photosensitization induced by the fluoroquinolone ofloxacin (OFLX) has been studied using two human cell lines, HL60 and K562, two UV wavelengths, 290 and 330 nm, and two different exposure protocols, acute and protracted. The examined endpoints are the cellular lethality and recovery and the membrane changes produced by the oxidative damage, studied using cloning and counting techniques and the measurement of the generalized polarization (GP) of the fluorescent membrane probe 2-dimethylamino-6-lauroyl-naphthalene (Laurdan). The results show that: (i) the photosensitizing effect is detectable at concentrations similar to those found in patients treated with OFLX only when the cells are irradiated with 330 nm; (ii) the amount of photodamage is a function of the drug concentration and of UV dose and persists also after the removal of the drug; (iii) during the first 24 h after OFLX treatment, a large decrease of the cell number can be observed due to cell lysis; (iv) the OFLX is inserted in the cell membranes at concentrations directly related with the drug concentration and incubation time; (v) the OFLX produces an increase in the GP values similar to that produced by membrane lipid oxidation which persists for hours after the removal of the drug. The overall results suggest the cell membrane as the main target of the OFLX adverse action, with a possible mechanism involving the formation of reactive oxygen species (ROS), which triggers, in turn, the lipid peroxidation chain reaction.

A subpopulation of low-density lipoproteins (LDL) is present in human plasma that contains lipid hydroperoxides and is more negatively charged (LDL−) than normal native LDL. By circular dichroism and tryptophan lifetime measurements we found that apoB-100 secondary structure is markedly decreased and its conformation is severely altered in LDL−. The low tryptophan fluorescence intensity confirms the oxidative degradation of the lipoprotein, and the very long lifetime value of one of its decay components indicates a low polarity environment for the remaining unbleached residues. Either a peculiar folding or, most likely, a sinking of the apoB-100 into the lipid core can account for the observed long lifetime component. Oxidation in vitro produces a similar unfolding of the apolipoprotein but the lifetime of tryptophan fluorescence is shifted to lower values, indicating that the denatured apoprotein remains at the hydrophilic surface of the lipoprotein particle. A disordering and an increased polarity of the LDL− surface lipids was demonstrated by measuring the generalized polarization of 2-dimethylamino-6-lauroylnaphthalene (Laurdan). The looser monolayer packing apparently favors the new conformation of apoB-100 and its sinking into a more hydrophobic environment, possibly accounting for it reduced receptor binding properties.