Figure 2.

Actin-based astrocyte extensions change orientation within hours after IOP elevation, in the presence or absence of viable axons. (A) Low-magnification images of the ONH and surrounding tissue labeled for actin filaments (red, TRITC-phalloidin), axonal tubulin (green, Tuj1 anti-tubulin βIII antibody), and nuclei (blue, DAPI). Superior (Sup.) and inferior (Inf.) anatomic locations are identified for orientation. The dashed box indicates the inferior ONH magnified in the top panel of (B). The arrow indicates the termination of Bruch's membrane (B, C) High-magnification images of the inferior ONH labeled for axonal tubulin, actin filaments, and nuclei. (B) Control ONH astrocytes display highly ordered actin-rich extensions, which are oriented perpendicular to the axonal and anterior (ant.)-posterior (post.) or A-P axis (top row). Immediately after CEI (Day 0), astrocyte extensions become disoriented relative to controls (middle row). Astrocyte extension reorient back to baseline orientation 1-day post-IOP normalization (CEI day 1; bottom row). (C) Two weeks post ONT, ONH axon tubulin labeling is essentially eliminated due to global optic nerve axon degeneration. However, ONH astrocyte extension maintain their orientation (top row) relative to controls (B, top row), despite total loss of axons. In post-ONT eyes, astrocyte extensions become immediately disoriented after CEI (C, CEI day 0 + ONT) relative to controls. Even post ONT, ONH astrocyte extensions reorient back to baseline orientation 1-day post-IOP normalization (C, CEI day 1 + ONT). (D) Mean astrocytic actin bundle orientation relative to the A-P axis within the ONH of control eyes and eyes exposed to ONT only, CEI only, or combined CEI + ONT. Error bars indicate standard error of the mean (SEM); *P < 0.05 and indicates a statistically significant difference between control and experimental groups; n = 7, 7, 7, 7, 6, and 4 for each column from left to right, and represents individual animals.