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Fig. 5. Normoxia induces differentiation of cultured retinal astrocytes. Cultures
of dissociated retinal cells from P1 animals were maintained for 4 days in
vitro at 20% oxygen and 1.5% oxygen. In situ hybridization revealed high
GFAP (A) and low VEGF mRNA levels in normoxic culture
conditions (C), and, conversely, low GFAP and high VEGF mRNA
levels in hypoxic conditions (B,D). Double immunolabeling (E,F) shows that the
GFAP-positive (red) cell population is identical to the Pax2-positive (green)
cell population after 24 (E) and 72 (F) hours in culture. (G) Assessment of
BrdU incorporation (red, inset) in GFAP-positive cells (green, inset) reveals
high proliferation in retinal astrocytes from P1 but a reduction in
proliferation over time in culture, whereas retinal astrocytes from P7 animals
were quiescent throughout the culture period. (H) The proliferation decrease
in P1 retinal astrocytes in culture is independent of the staining method
(anti-GFAP or -Pax2 labeling) used to identify retinal astrocytes. (I)
However, culturing P1 retinal cells in 1.5% O2 largely prevents the
decline in astrocyte proliferation seen under normoxic conditions. Scale bars:
50 µm.
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