Abstract
Purpose :
We previously reported that fasudil, a Rho-Kinase II inhibitor, reduces optic nerve axonal injury in rats following a Controlled Elevation of Intraocular Pressure (CEI). Work is in progress to determine the mechanisms of protection. Here, we investigate the impact that fasudil has on astrocyte proliferation within the optic nerve head (ONH) following CEI.
Methods :
Adult male Brown Norway rats (n=28) were injected intraperitoneally with either fasudil (10 mg/kg/day; n=12) or vehicle (water; n=16) the day before an 8 hour CEI, and then daily until they were euthanized. Animals were euthanized immediately after CEI (0 day; n=12) or 3 days (n=16) following CEI. Following perfusion with 4% paraformaldehyde, globes were cryo-sectioned longitudinally. Proliferating astrocytes were identified by co-immunolabelling of Ki67 and Sox2, proliferation and astrocytic-nuclear markers, respectively. Nuclear counting was performed in the region 150 μm posterior to Bruch’s membrane. Nuclear cell densities were compared between fasudil and vehicle treated animals, as well as between CEI and contralateral eyes, using ANOVA and mixed effect models.
Results :
Astrocyte densities in CEI ONHs were significantly lower than contralateral ONH in both fasudil (p=0.002 at 0 day and p=0.0002 at 3 day) and vehicle (p=0.005 at 3 day) groups. As expected, there was minimal (<1%) ONH proliferation immediately following CEI (0 day). On day 3, vehicle treated ONHs had a significantly higher number of proliferating cells (189 ± 112 nuclei/mm2) than fasudil treated animals (32 ± 29 nuclei/mm2; p=0.008). Proliferating nuclei (Ki67+) accounted for 1% (fasudil) vs 8% (vehicle) of all ONH nuclei (DAPI). Between 70% (vehicle) to 100% (fasudil) of these proliferating nuclei were astrocytes (Sox2+). Fasudil treated animals had fewer proliferating astrocytes (Ki67 + Sox2; p=0.03) than water treated animals following CEI at 3 days.
Conclusions :
Our results support the hypothesis that fasudil suppresses optic nerve head astrocyte proliferation following an induced glaucomatous event, and that astrocyte proliferation may be deleterious to ONH axonal health. RNA analyses are underway to determine ONH gene regulation with fasudil treatment following CEI.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.