Fig. 6

Astrocytes rendered reactive by Hp OMVs promote neuronal damage. a Experimental design to test the effect of (1) the plasma membrane of reactive astrocytes on neuronal differentiation, and (2) Hp OMVs on differentiated neurons. (1) Astrocytes were treated (with or without TNF or OMVs) and then fixed. CAD cells labeled with cell tracker green were cultured on a plate (control for CAD differentiation) or seeded and co-cultured on the fixed astrocyte monolayer for 24 h. (2) CAD cells differentiated for 48 h were treated with OMVs (2.5 μg/ml, 24 h) to explore the effect of OMVs on neuronal process retraction and cell viability. CAD cell morphological differentiation was estimated by measuring the length of neuronal processes using the NeuronJ plug-in of the NIH ImageJ Software. b Confocal microscopy of CAD cells (stained with cell tracker green) under the different experimental conditions. White arrows indicate neurites that extended on the tissue culture plate (plate), over untreated astrocytes (Control), or astrocytes treated with TNF or with OMVs (scale bar = 20 µm). c Quantification of neurite length (μm). The values in the graph are the means ± S.E.M.; n = 4, and **p < 0.01. We measured the neurite length of 50 neurons per condition, per experiment. d Phase-contrast microphotographs of CAD cells differentiated on the tissue culture plate without treatment (Control) or treated with OMVs (OMVs). Yellow arrowheads indicate neurites that extended on the tissue culture plate (scale bar = 20 µm). e Quantification of neurite length (µm) obtained from the images, as shown in d. Values are means ± S.E.M.; n = 4, and **p < 0.01. f MTS assay of differentiated CAD cells treated or not (Control) with OMVs for 12 or 24 h. The graph shows the percentage of viable cells