Koshu GSE augments cell survival of cultured hippocampal neurons after excitotoxic treatment.

<p>Hippocampal neurons plated at 2.0×10⁵ cells/cm² were treated with 50 µM glutamate in the presence or absence of 1.0 ng/ml KOS GSE for 10 min and allowed to recover in conditioned medium for 24 hr. (A) Representative images of hippocampal neurons after treatment. Bar, 100 µm. (B) The mean density of MAP2-positive neurons surviving 24 hr after glutamate treatment (<i>n</i> = 8). ***, <i>P</i><0.001; *, <i>P</i><0.05. (C) Quantification of the mean dendrite length by morphometric analysis (<i>n</i> = 8). ***, <i>P</i><0.001; *, <i>P</i><0.05.</p

Effect of Koshu GSE on active caspase-3.

<p>(A) Hippocampal neurons treated with 50 µM glutamate alone or in the presence of the indicated concentrations (in ng/ml) of KOS GSE for 30 min were analyzed for active-caspase-3 by Western blot. Numbers below the active caspase-3 bands indicate their relative intensities quantified by densitometric analysis. (B) Hippocampal neurons treated with 50 µM glutamate in the presence or absence of 1.0 ng/ml KOS GSE for 30 min and incubated in normal culture medium for 0, 3, or 6 hr were analyzed for active-caspase-3 by Western blot. Numbers below the active caspase-3 bands indicate their relative intensities quantified by densitometric analysis.</p

Typical liquid chromatography profile of Koshu and MBA grape seed extracts.

<p>Polyphenols in the eluate were detected by absorbance at 280 nm. The peaks were assigned by TOF-MS as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014575#pone-0014575-t001" target="_blank">Table 1</a>.</p

LC/TOF-MS data of phenolic compounds.

<p>Asterisk indicates those only detected in KOS GSE.</p

Koshu GSE alleviates glutamate-induced inactivation of Erk1/2 in cultured hippocampal neurons.

<p>(A) <i>Top</i>, detection of phosphorylated Erk1/2 in cultured hippocampal neurons by western blot. Neurons were treated with 50 µM glutamate alone or in the presence of indicated concentrations (in ng/ml) of KOS or MBA for 30 min. The immunoblot for total Erk1/2 shows equal loading. <i>Bottom</i>, quantification of phospho-Erk1/2 levels by image analysis (<i>n</i> = 5). The relative values to the untreated controls (set to 100%) are shown. *, <i>P</i><0.05 versus untreated controls. (B) Hippocampal neurons were treated with 50 µM glutamate with or without indicated concentrations (in ng/ml) of KOS GSE for 30 min, and 200 µg of total protein were loaded in each lane for western blot analyses for phospho- and total Akt. The basal Akt phosphorylation appeared slightly diminished upon glutamate insult. KOS GSE did not show any protective effect on basal Akt phosphorylation levels. (C) The effect of KOS GSE alone on Erk1/2 phosphorylation in cultured hippocampal neurons. Cells were treated with indicated concentrations (in ng/ml) of KOS GSE for 30 min. <i>Top</i>, representative western blot images of phospho- and total-Erk1/2. <i>Bottom</i>, a quantification of the phospho-Erk1/2 levels by image analysis (<i>n</i> = 3).</p

Koshu GSE protects dendrite processing of cultured hippocampal neurons exposed to a toxic concentration of glutamate.

<p>(A) Representative image of untreated hippocampal neurons at 8 DIV immunostained for MAP2. Bar, 100 µm. (B) Representative binary images of cultured hippocampal neurons immunostained for MAP2 with no treatment or after a 30 min treatment with 50 µM glutamate alone, 50 µM glutamate plus 1.0 ng/ml KOS, or 50 µM glutamate plus 1.0 ng/ml MBA. Bar, 100 µm. (C) Quantification of the mean dendrite length by morphometric analysis (<i>n</i> = 10). ***, <i>P</i><0.001 versus untreated controls.</p