Cell age-dependent Glu-uptake and the effects of EPO on the expression of GLAST by astroglial primary cultures (APC) under normoxic and hypoxic culture conditions.

<p>(<b>A</b>) Quantification of GLAST-positive astrocytes (GLAST+/GFAP+cells) under normoxia shows a significant (**p<0.01; ***p<0.001) increase in GLAST+/GFAP+cells upon EPO-treatment in both young and aged APC (D7 and D21); (<b>B</b>) hypoxia enhanced the effect of EPO on GLAST/GFAP+ cells. In both young and aged cells the number of GLAST/GFAP cells was increased (**p<0.01 at d7 and ***p<0.001 at d21). (<b>C</b>) The uptake of 1 mM glutamate by APC (shown in absolute values) was increased on day 14 in comparison with day 7 under normoxia (white bars, **p<0.01) and hypoxia (grey bars, ***p<0.001) and strongly decreased in 21-day old cultures (d21) exposed to hypoxia (grey bars) when compared to those from day 7 in hypoxia (grey bars d21vs. d7, p***<0.001).</p

The significance of EPOR for astroglial cell survival and utilization of glutamate by astroglial cells.

<p>(<b>A</b>) Immunostaining for EPOR (green) and β-tubulin III (red) in DIV14 APC; (<b>B</b>) Immunostaining of EPOR (green) and GFAP (red) of DIV14 APC; (<b>C</b>) Immunostaining for EPOR (green) and /ß-tubulin III (red) in DIV14 APC transfected with EPOR siRNA; (<b>D</b>) Immunostaining of and EPOR (green) and /GFAP (red) of 14-day-old APC transfected with EPOR siRNA. Cell nuclei are stained with DAPI shown in blue. Scale bar 200µm. (<b>E</b>) Caspase 3/7 activity in untreated 14-day-old APC (white bars) and those transfected with siRNA for EPOR upon normoxia (patterned white bars) (<b>F</b>) Caspase 3/7 activity in hypoxic untreated 14-day-old APC (grey bars) and those transfected with siRNA for EPOR (patterned grey bars) Treatment of original APC (EPOR+) with transfection reagent (TR) did not influence significantly the survival of APC under both normoxic (white bars) and hypoxic (grey bars) conditions. (<b>G</b>) Glu uptake in untreated (EPOR+) APC at DIV14 and those transfected with EPOR siRNA (EPOR-) under normoxia (white bars) and hypoxia (grey bars) (<b>H</b>) GS-activity in untreated (EPOR+) APC at DIV14 and those transfected with EPOR siRNA (EPOR-) under normoxia (white bars) and hypoxia (grey bars). Treatment of original APC (EPOR+) with transfection reagent (contr TR) did not influence significantly the GS-activity of APC. * p<0.05, ** p<0.01, *** p<0.001.</p

Cell age- and concentration-dependent effects of EPO on glutamine synthetase activity.

<p>(<b>A</b>) DIV7 APC under normoxia treated with (+1mM Glu) or without (-Glu) and 1 or 5U/ml EPO (1UE or 5 UE respectively); (<b>B</b>) DIV7 APC under 24h normoxia /white bars) or hypoxia (grey bars) treated with (+Glu) or without (-Glu) and 1 or 5U/ml EPO (1UE or 5 UE respectively); (<b>C</b>) DIV14 APC under normoxia treated with (+Glu) or without (-Glu) and 1 or 5U/ml EPO; (<b>D</b>) DIV14 APC upon 24h normoxia /white bars) or hypoxia (grey bars) treated with (+Glu) or without (-Glu) and 1 or 5U/ml EPO (1UE or 5 UE respectively); (<b>E</b>) DIV21 APC under normoxia treated with (+Glu) or without (-Glu) and 1 or 5U/ml EPO (1UE or 5 UE respectively); (<b>F</b>) DIV21 APC under 24h normoxia /white bars) or hypoxia (grey bars) treated with (+Glu) or without (-Glu) and 1 or 5U/ml EPO (1UE or 5 UE respectively). At all three time points in culture, Glu increased the activity of GS when compared to respective controls culture without Glu (-Glu). Treatment with EPO increased the glutamate-induced activation of GS in concentration-dependent manner when compared to control culture (cf. +Glu vs +Glu+1U E and +Glu+5U E). *, p<0.05, **, p<0.01, ***, p<0.001.</p

Expression of EPOR/GFAP in young and culture aged APC under normoxia and hypoxia.

<p>(<b>A</b>) Combined picture of double immunostaining of EPOR (green) and GFAP (red) in 7-day old APC under normoxia; (<b>B</b>) Corresponding picture of EPOR in green; (<b>C</b>) Double immunostaining of EPOR (green) and GFAP (red) of APC DIV 7 upon hypoxia; (<b>D</b>) Corresponding picture to C of EPOR (green) only; (<b>E</b>) EPOR (green) and GFAP (red) expression in APC on DIV21 under normoxia; (<b>F</b>) EPOR (green) picture corresponding to E; (<b>G</b>) EPOR(green) and GFAP(red) expression in APC at DIV21 upon hypoxia; (<b>H</b>) corresponding to G picture of EPOR (green) only in DIV21 APC under hypoxia. A-H Nuclear staining with DAPI shown in blue, scale bar 200µm.</p

Cell age-dependent effects of glutamate, hypoxia on the expression of EPO, glutamine synthetase (GS), EPO release and EPO mRNA expression in rat APC.

<p>(<b>A</b>) Western blot analysis of GS and EPO in cell homogenates prepared from 7- (DIV7) and 21-day-old (DIV21) rat APC under normal and hypoxic conditions with or without exposure to Glu and EPO. (<b>B</b>) The cell-age-dependent release of EPO into the culture medium under normoxia (N, white bars) and 24h or 48h hypoxia (H, grey bars) in 7-,15- and 21-day old APC. (<b>C</b>) The effect of EPO treatment (5U/ml) on the expression of EPO mRNA in early (7-day-old) APC under normoxia (N, white bars) and hypoxia (H, grey bars). (<b>D</b>) The effect of EPO on the expression of EPO mRNA in prolonged (21-day-old) APC under normoxic (N) and hypoxic (H) culture conditions. Data are normalized to normoxic control (N) *, p<0.05, **, p<0.01, ***, p<0.001.</p

Quantification of GFAP/PCNA-positive cells in APC in young and culture aged APC upon hypoxia/Glu/EPO-exposure.

<p>(<b>A</b>) GFAP/PCNA+ cell counts in APC at DIV7 exposed to Glu under normoxia (N, white bars) and hypoxia (H, grey bars) showed an increase of proliferating astrocytes upon exposure to 5U/ml EPO (5U E) only under hypoxic conditions. Hypoxia decreased the proliferation of APC (cf- N+Glu vs. H+Gu); (<b>B</b>) Quantification of GFAP/PCNA+ cells on DIV21 shows no changes in PCNA+ astrocytes exposed to EPO under normoxia (white bars), while a significant increase (**p<0.01) is detected in EPO-treated APC (H+Glu+5U E) under hypoxia (grey bars), as compared with the hypoxic control (H+Glu). The proliferation of APC in normoxic (N+Glu) and hypoxic control conditions (H+Glu) remained unchanged.</p