Abstract
Purpose :
To investigate the effects of topical ripasudil, a Rho kinase (ROCK) inhibitor, to mouse optic using two independent models.
Methods :
Young CD1 mice(N=122) received daily saline(BSS) or 2% ripasudil(2%R) eyedrops, starting 3 days prior to microbead injection(GL), with intraocular pressure(IOP) elevation for 1d or 3d, or 6wk (total=6 or 8 doses). Additional mice underwent optic nerve crush with either BSS or 2%R drops(2 week survival; n=20). Others received only 2%R drops(N=43) or were bilaterally naïve(BN)(N=38). Axonal transport of amyloid precursor protein(APP) was assessed quantitatively with immunofluorescence(method previously published). Axon loss was estimated in epoxy sections of optic nerve(ON) & by counting RBPMS+ ganglion cells(RGC) in retina whole mounts. Other eyes were microdissected by region: retina, unmyelinated optic nerve(UON) & myelinated optic nerve(MON). Regional protein expression for ROCK pathway molecules was quantified by micro-western analysis(WES) with GAPDH control after 3 days GL & 2%R or BSS drops.
Results :
2%R lowered IOP at 4 hours(p≤0.001), but IOP was normal at 1 day & 2%R did not affect IOP increase with GL. Axonal transport block was seen in both BSS & 2%R GL groups(p<0.05). Axonal transport was maintained in fellow non-GL 2%R eyes but reduced in fellow BSS+GL eyes(p<0.05). Axon loss at 6 weeks was significantly less in 2%R+GL MON(8% vs 33% in BSS+GL; p=0.02). Likewise, RGC loss was less with 2%R vs BSS after crush(68% vs 80%; p=0.005). 2%R alone had no effect on RGC count. By Wes(Figure), 2%R+GL UON had significantly higher cofilin, ROCK2, p-ROCK2, & p38 than BN(p<0.05), while BSS+GL did not(all p≥0.1). In MON but not UON, both GL groups had increases in ROCK2, p38, & RhoA. In MON, pROCK2 was increased in 2%R+GL & p-cofilin was increased in BSS+GL. Further studies of ROCK activity & UON biomechanics are ongoing.
Conclusions :
Topical 2%R reduced RGC loss in both GL & crush models, maintained median axonal transport of APP, & differentially affected members of the ROCK pathway in regions of the ON. Neuroprotective effects of ripasudil may have multiple sites of action.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.