Rho kinase inhibition promotes stellation in primary cultured optic nerve head astrocytes

Abstract

Purpose : Astrocytes are stellate-shaped glia that support optic nerve head (ONH) axons with numerous actin-rich processes. With intraocular pressure (IOP) elevation in animal models, ONH astrocytes retract their processes. Here, we determined if Rho kinase inhibition with fasudil promotes stellation in cultured primary ONH astrocytes.

Methods : Adult rat ONH astrocytes were collected and validated as previously described. Cultures of passages 10-15 were seeded at 25,000 cells/cm². After 24h, cells were treated with fasudil (0, 1, 5, 10, 25, 50, and 100µM) for an additional 24h. Cells were fixed in 4% paraformaldehyde solution, labeled for actin and nuclei, and imaged using confocal microscopy. MTT uptake and LDH release assays were performed to determine any cytotoxic effects from fasudil. Cell shape and Sholl-like analyses (5-170µm radii circles from the cell center) were used to determine cell area and perimeter, cell circularity (graded on a scale of 0-1, where 1 represents a circle), and mean/maximum process lengths, respectively, via Adobe Photoshop. Data were analyzed using ANOVA statistical analysis (n=53±14 cells/group) and presented as mean ± SEM.

Results : Control astrocytes were non-stellate in shape. Mean cell area and cell viability remained unchanged with 1-100µM fasudil treatment. With increasing fasudil concentration, cells became more stellate in shape. Mean cell perimeter increased with fasudil treatment (significant at fasudil concentrations ≥50µM: 377.9±14.3µm in experimental vs. 293.4±12.0 in controls; p=0.001). Mean cell circularity decreased with fasudil treatment (significant at fasudil concentrations ≥10µM: 0.24±0.01 in experimental vs. 0.31±0.01 in controls; p=0.001). In Sholl-like analyses, mean astrocyte process number of intersections at various radii increased with fasudil treatment (most intersections at 25µm from the cell center; significant at fasudil concentrations ≥50µM: 3.7±0.2 in experimental vs. 3.0±0.1 in controls; p=0.03). Mean maximum cell process length increased with increasing fasudil concentrations (by up to 1.4x in experimental vs. control cells; linear regression slope=3.5, r²=0.63, p<0.0001).

Conclusions : Rho kinase inhibition promotes stellation in cultured ONH astrocytes, mirroring astrocyte morphology described in ONH tissue. Rho kinase inhibition in vivo may maintain astrocyte stellate morphology in the setting of elevated IOP.

This is a 2021 ARVO Annual Meeting abstract.