Effects of topical Rho kinase inhibitor, ripasudil, on murine optic nerve astrocytes in a glaucoma model

Abstract

Purpose : To assess effects of ripasudil(R) on optic nerve(ON) astrocytes in a mouse glaucoma(GL) model.

Methods : Young CD1 mice(N=37) received 6 doses of daily saline(BSS) or 2%R eyedrops starting 3 days before GL induction by microbead injection, then followed for 3 days(N=15) or 6 weeks(N=10), versus untreated control eyes(N=8) & 2%R drops only(N=4). Cross-sections at pre-lamina, unmyelinated ON(UON), & myelinated ON(MON) were labeled for glial fibrillary acidic protein(GFAP) & DAPI to quantify astrocyte process structure and nuclei as well as Rho kinase(ROCK)-associated proteins: RhoA, p38, cofilin, ROCK2 & phosphorylated-ROCK2.

Results : Control astrocyte processes were thicker at the ON periphery & occupied 50% of total ON area. In 3 day GL, ON area increased(p≤0.04) & GFAP area fraction decreased 4-8%(ns) in both GL groups. Mean astrocyte process width was wider only in 6 week GL 2%R vs BSS & naïve(p≤0.004, Fig1). With 3 day GL in both groups, a significant 2.5X increase occurred in nuclei area(both GL ≤0.002) & nuclei number(p≤0.003), which largely returned to control levels by 6 weeks. The nuclear aspect ratio parameter was less oval in BSS GL at 3 days & 6 weeks than control(p≤0.02), while 2%R GL groups had insignificant change. 2%R did not reduce the intraocular pressure rise in GL eyes. 2%R alone reduced the area occupied by nuclei compared to all other groups(p≤0.04). Axons & astrocytes of GL MON have increased RhoA labeling over controls. p-ROCK2 stains axons most intensely in the GL MON. Cofilin(Fig 1C,D) & p38 are labeled more strongly in astrocytes than axons in GL UON & MON tissue. P-cofilin stains GL & control tissue similarly. ROCK2 stains axons in both control & GL MON.

Conclusions : Microbead GL alters mouse astrocyte processes, orientation, & nuclear density in a pattern compatible with initial reorientation & partial reversibility of GL changes. Topical 2%R differentially mediates some effects and is shown to be neuroprotective in parallel experiments.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Figure1. The 6 week GL 2%R UON(B) mean astrocyte process width was thicker than naïve UON(A, green- GFAP, red-phalloidin, blue-DAPI). UON stained with cofilin(green) shifted from staining both the astrocytes & axons in control(C) to astrocytes only in 3 day GL UON(D).