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Shandiz Tehrani, Elaine C Johnson, William O Cepurna, John C Morrison; Actin-Rich Astrocytic Process Orientation Delineates the Optic Nerve Head and Signifies a Potential Marker of Physiologic Stress in the Optic Nerve. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2403.
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© ARVO (1962-2015); The Authors (2016-present)
Optic nerve head (ONH) astrocytes may sense stress even in the setting of normal intraocular pressure (IOP). Sensitive markers of astrocytic process arrangement may provide insights into subtle stresses within the ONH. This study was designed to identify and quantify the intensity and orientation of astrocytic processes at different levels of the normal rat optic nerve (ON) using the filamentous actin marker, phalloidin.
Longitudinal sections of rat ON (n=4) were co-labeled with phalloidin and antibodies to astrocytic glial fibrillary acidic protein (GFAP) or axonal tubulin βIII (Tuj1). Confocal microscopy imaging and FIJI analysis were used to quantify the intensity and orientation of actin or GFAP labeled filaments at different regions of the ONH and ON, relative to Bruch’s membrane (BM).
Phalloidin labeling showed densely arranged actin filaments within the ONH and ON that localized parallel to GFAP positivity, suggesting that actin-rich astrocytic processes may be the dominant source of actin filaments (Figures 1A-B). Actin filament orientation within the ONH was nearly perpendicular to axons (82.9° ± 6.3° relative to axonal axis) (Figures 1C, 2A). More than 250µm posterior to BM, actin filament orientation became significantly more random (45.4° ± 28.7° relative to axonal axis, ANOVA p<0.05) (Figures 1D, 2B-C). GFAP-labeled filaments in the ONH mirrored actin filament orientation, but with a larger distribution (73.6° ± 20.0° relative to axonal axis). While there was a trend toward more random GFAP-labeled filament orientation posterior to BM, this was not statistically significant (Figure 2C). Actin or GFAP fluorescence labeling intensity did not significantly change along the ON (Figure 2D).
Actin filaments in the ONH are uniquely ordered relative to axons, in contrast with actin filaments within the remainder of the ON. We hypothesize that actin filament labeling with phalloidin is indicative of fine astrocytic processes not reflected by GFAP. The difference in ONH astrocytic process orientation may be the result of normal physiologic stresses unique to the ONH, and this orientation may be a sensitive marker for astrocyte response to elevated IOP.
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