Purpose
To assess the structure and cellular origin of the filamentous actin (F-actin) optic nerve head (ONH) cytoskeleton at normal and elevated intraocular pressure (IOP) in the rat.
Methods
Unilateral IOP elevation was produced in rats by episcleral injection of hypertonic saline and tissues collected at 5 weeks. For comparison, ONH following optic nerve transection were generated. Optic nerve axonal degeneration was graded on a scale of 1(normal)-5 (extensive) by light microscopy. Longitudinal sections of rat ONH were colabeled with phalloidin and antibodies to astrocytic aquaporin (Aqp4), or axonal tubulin βIII (Tuj1) to further elucidate the cellular origin of F-actin and its relationship to axons.
Results
Untreated rat ONH showed densely arranged F-actin bundles oriented perpendicular to the vertical axis, and to Tuj1 labeled axons (not shown), in a pattern most consistent with the orientation of astrocytic processes in the glial lamina (Figure 1a). Additionally, F-actin labeled the walls of the ONH vascular components. F-actin partially co-labeled with Aqp4, further suggesting the astrocytic origin of the dense F-actin bundles (Figure 2). Preliminary labeling intensity analysis of 8 glaucoma model eyes with axonal injury grades from 1 to 4.9 (mean and peak IOP±SD from 19.2±1.6 to 29.6±3.9 and 22±3.2 to 50.4±1.3 mmHg, respectively) suggested that astrocytic structural changes, as indicated by reduced F-actin labeling in eyes with minimal injury (Figure 1b), may precede axonal degeneration. With greater injury (Figure 1c), the ONH actin cytoskeleton became exceedingly disorganized with an apparent increase in F-actin fluorescence intensity, possibly due to astrocytic remodeling in response to injury. In contrast, in transected ONH, F-actin remained relatively preserved, suggesting that the changes in the F-actin cytoskeleton with IOP elevation were not simply secondary to non-specific axonal injury.
Conclusions
The actin cytoskeleton of the rat ONH is a dense structure that may have a role in early and late ONH remodeling in response to glaucomatous injury. F-actin labeling within the ONH appears to highlight the glial lamina more effectively when compared to Aqp4 labeling. In addition, F-actin labeling highlights the vascular components of the ONH.
Keywords: 629 optic nerve •
430 astrocytes: optic nerve head •
493 cytoskeleton