Astrocyte Processes Label for Filamentous Actin and Reorient Early Within the Optic Nerve Head in a Rat Glaucoma Model

PURPOSE. To determine if astrocyte processes label for actin and to quantify the orientation of astrocytic processes within the optic nerve head (ONH) in a rat glaucoma model. METHODS. Chronic intraocular pressure (IOP) elevation was produced by episcleral hypertonic saline injection and tissues were collected after 5 weeks. For comparison, eyes with optic nerve transection were collected at 2 weeks. Fellow eyes served as controls. Axonal degeneration in retrobulbar optic nerves was graded on a scale of 1 to 5. Optic nerve head sections (n > 4 eyes per group) were colabeled with phalloidin (actin marker) and antibodies to astrocytic glial fibrillary acidic protein and aquaporin 4, or axonal tubulin beta III. Confocal microscopy and FIJI software were used to quantify the orientation of actin bundles. RESULTS. Control ONHs showed stereotypically arranged actin bundles within astrocyte processes. Optic nerve head actin bundle orientation was nearly perpendicular to axons (82.9 degrees +/- 6.3 degrees relative to axonal axis), unlike the retrobulbar optic nerve (45.4 degrees +/- 28.7 degrees, P < 0.05). With IOP elevation, ONH actin bundle orientation became less perpendicular to axons, even in eyes with no perceivable axonal injury (i.e., 38.8 degrees +/- 15.1 degrees in grade 1, P < 0.05 in comparison to control ONHs). With severe injury, ONH actin bundle orientation became more parallel to the axonal axis (24.1 degrees +/- 28.4 degrees degrees, P < 0.05 in comparison to control ONHs). Optic nerve head actin bundle orientation in transected optic nerves was unchanged. CONCLUSIONS. Actin labeling identifies fine astrocyte processes within the ONH. Optic nerve head astrocyte process reorientation occurs early in response to elevated IOP.