12 research outputs found

    Effect of the PLL coated surfaces on neurite elongation and cellular polarization.

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    <p><b>A</b>. Fluorescently labeled cortical neurons, immunostained for TAU (green); nuclei are counterstainned with Hoechst (blue); <b>B.</b> Number of primary neurites per cell; <b>C.</b> Total neurite length; <b>D.</b> Average neurite length and <b>E.</b> Length of the longest neurite. (n = 130 cells, mean ± SD, *** for p<0.001).</p

    Cortical neuron culture on PLL coated films of P(TMC-CL) and respective homopolymers (2.7×10<sup>4</sup> viable cells were seeded per sample).

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    <p><b>A.</b> Number of cortical neurons with and without neurite extensions on polymeric surfaces coated with aqueous solutions at different concentrations of PLL. Glass coated with 24 µg.µl<sup>−1</sup> of PLL for 30 minutes was used as control. (n = 3 independent studies, mean ± SD, p<0.05) <b>B.</b> Percentage of PLL covered surface area as a function of the coating conditions. (n = 3, mean ± SD, p<0.05). x = condition not tested, 0 = null value. n.s. = non-significantly different from the control, α = total number of cells not significantly different from the control, β = number of cells with neurite extensions not significantly different from the control and χ = number of cells without extensions not significantly different from the control.</p

    Analysis of GSK3β in cortical neurons plated on P(TMC-CL) and effects of GSK3β inhibition on neurite extension.

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    <p><b>A.</b> Schematic representation of the different phosphorylation forms of GSK3β and their activity status; <b>B.</b> Analysis of the phosphorylated forms of GSK3β by western blot. Representative blots are shown. Expression levels of GSK3β isoforms, β1 and β2, are presented and quantified individually or together. (n = 3 independent studies, average ± SD); <b>C.</b> Morphology of neurons (immunostained for TAU in green and nuclei counterstained in blue) cultured for 24 hours in the presence of DMSO (control) or in the presence of 6-bromoindirubin-3′-acetoxime (BIO) at 30 and 300 nM. Quantifications of the longest neurite, average neurite length and the number of neurites per cell are shown (n = 130 cells, mean ± SD, * for p<0.05, ** for p<0.01 and *** for p<0.001); <b>D.</b> Determination of CRMP4 phosphorylation levels in cortical neurons plated for 4 days on control or P(TMC-CL). Representative western blot is shown and below the quantification (n = 3 independent studies, average ± SD).</p

    Morphology and mechanical properties of the tested polymeric surfaces.

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    <p><b>A</b>. Root mean square (RMS) roughness of all polymeric surfaces; <b>B</b>. Representative photographs of the polymeric surfaces before and after nanoindentation; images are color coded, showing elevated areas in bright and lower areas in dark color. <b>C</b>. Representative nanoindentation force-displacement curves; D. Mean hardness values of all polymeric surfaces, calculated for the maximum load and E. Mean stiffness values for all polymeric surfaces. (n = 60 indentations, mean ± SD, *** for p<0.001).</p

    Characteristics of the synthesized and purified P(TMC-CL) (co)polymers.

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    a)<p>Determined by 1H NMR on specimens purified by precipitation;</p>b)<p>Determined by GPC at 30°C using chloroform as the eluent.</p

    Effect of CNS myelin on neurite outgrowth of cortical neurons cultured for 4 days on PLL-P(TMC-CL) substrates coated with CNS myelin.

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    <p><b>A.</b> Cortical neurons are immunostained for β-III tubulin (green) and nuclei are counterstainned with Hoechst (blue); myelin coating is immunostainned for MBP (green), surfaces were fully covered by myelin (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088593#pone.0088593.s002" target="_blank">fig. S2</a> for myelin quantification) <b>B.</b> Effect of myelin on the ability of neurons to extend processes is presented as the % of cells with neurites in relation to the total number of cells. (n = 3 independent studies, mean ± SD; ** for p<0.01).</p

    Plasmalogen levels after AG treatment in young mice.

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    <p>Plasmalogen levels in several tissues from 20-days old (P20) mice fed a control diet (WT pups n = 4; <i>Pex7</i> KO pups n = 4) or an AG diet (WT pups n = 5; <i>Pex7</i> KO pups n = 6). Increased levels of plasmalogens are observed in kidney, testis, eye and intestine.</p

    Nerve conduction improves after AG treatment.

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    <p>(A) Representative examples of compound muscle action potentials recordings after stimulation at the sciatic notch of wild type and <i>Pex7</i> KO mice fed control or AG diets. Increased latencies were observed in control-fed <i>Pex7</i> KO mice that were partially restored after the AG diet. (F) Calculated motor nerve conduction velocities (MNCV) of wild type and <i>Pex7</i> KO mice fed control or AG diets. Bars represent the average values obtained after bilateral measurements in wild type and <i>Pex7</i> KO mice, and the significance is shown above each comparison. The AG diet partially restores MNCV in sciatic nerves of <i>Pex7</i> KO mice. Numbers of mice analyzed: on control diet WT mice n = 4 and <i>Pex7</i> KO mice n = 3, on AG diet WT mice n = 4 and <i>Pex7</i> KO mice n = 4.</p

    Deficiency in plasmalogens affects lipid droplet formation in MEFs.

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    <p>(A) Lipid droplets were stained with BODIPY 493/503 in MEFs from WT and Gnpat KO mice (n = 4 per genotype). MEFs were cultured in normal medium (upper panels), in control medium (bottom left panel) or in medium supplemented with 15 µM AG (bottom right panel). In MEFs from Gnpat KO mice, the deficiency in plasmalogens leads to a reduction in the number and volume of lipid droplets (B, C). Treated of Gnpat KO MEFs with 15 µM AG for 7 days lead to an increase in the number and volume of lipid droplets (B, C) to values similar to those of lipid droplets from WT MEFs. * p<0.02 using Mann Whitney test.</p

    AG treatment in young mice prevents tissue pathology.

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    <p>(<b>A</b>) Testis sections of P20 WT and <i>Pex7</i> KO pups stained with hematoxylin and eosin (H&E). Whereas control-fed <i>Pex7</i> KO pups already showed the degenerative changes in spermatocytes, AG-fed <i>Pex7</i> KO pups where protected from degeneration and showed normal appearing seminiferous tubules and spermatocytes. Bars are 50 µm. (B) Eye sections of P20 WT and <i>Pex7</i> KO pups stained with Richardson's stain. In contrast to the lens of WT pups showing organized and orientated fiber cells, the cataract in the lens of control-fed <i>Pex7</i> KO pups shows abnormally sized and abnormally arranged fiber cells. The AG diet prevented the abnormal development of fiber cells in <i>Pex7</i> KO pups thus preventing cataract formation. Bars are 50 µm. (C) Eye sections of mice showing the correlation between plasmalogen (Pls) levels and the development of cataracts in <i>Pex7</i> KO pups (from each mouse one eye was used for histology and the other eye used for biochemical analyses). Whereas complete loss of plasmalogens (Pls = 0.0) leads to a massive cataract occupying the entire lens (the cataract area is circled with a dashed line), a plasmalogen level of 2.7% is able to prevent cataract formation in <i>Pex7</i> KO pups. Partial restoration of plasmalogens (Pls = 1.2%) leads a small nuclear cataract (circled with a dashed line). Sections were stained as in (B) and bars are 100 µm. Numbers of mice analyzed: on control diet WT mice n = 4 and <i>Pex7</i> KO mice n = 4, on AG diet WT mice n = 4 and <i>Pex7</i> KO mice n = 6.</p
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