The Mechanisms of Neuroprotection by Topical Rho Kinase Inhibition in Experimental Mouse Glaucoma and Optic Neuropathy

• Published in: Investigative ophthalmology & visual science 2024-11, Vol.65 (13), p.43
• Main Authors: Quillen, Sarah E, Kimball, Elizabeth C, Ritter-Gordy, Kelsey A, Du, Liya, Yuan, Zhuochen, Pease, Mary E, Madhoun, Salaheddine, Nguyen, Thao D, Johnson, Thomas V, Quigley, Harry A, Pitha, Ian F
• Format: Article
• Language: English
• Subjects: Administration, Topical; Animals; Astrocytes - drug effects; Astrocytes - metabolism; Axonal Transport - drug effects; Blotting, Western; Disease Models, Animal; Glaucoma - drug therapy; Glaucoma - metabolism; Intraocular Pressure - drug effects; Intraocular Pressure - physiology; Isoquinolines - administration & dosage; Isoquinolines - pharmacology; Male; Mice; Mice, Inbred C57BL; Neuroprotection - drug effects; Neuroprotective Agents - administration & dosage; Ophthalmic Solutions; Optic Nerve Diseases - drug therapy; Optic Nerve Diseases - etiology; Optic Nerve Diseases - metabolism; Optic Nerve Diseases - prevention & control; Retinal Ganglion Cells - drug effects; Retinal Ganglion Cells - metabolism; Retinal Ganglion Cells - pathology; rho-Associated Kinases - antagonists & inhibitors; Sulfonamides - administration & dosage; Sulfonamides - pharmacology
• ISSN: 1552-5783; 1552-5783
• DOI: 10.1167/iovs.65.13.43

The purpose of this study was to delineate the neuroprotective mechanisms of topical 2% ripasudil (Rip), a Rho kinase (ROCK) inhibitor. In 340 mice, scheduled 2% Rip or balanced salt solution (BSS) saline drops were intermittently, unilaterally delivered. Intracameral microbead glaucoma (GL) injection increased intraocular pressure (IOP) from 1 day to 6 weeks (6W), whereas other mice underwent optic nerve (ON) crush. Retinal ganglion cell (RGC) loss was assessed using retinal wholemount anti-RNA Binding Protein with Multiple Splicing (RBPMS) labeling and ON axon counts. Axonal transport was quantified with β-amyloid precursor protein (APP) immunolocalization. Micro-Western (Wes) analysis quantified protein expression. Immunofluorescent expression of ROCK pathway molecules, quantitative astrocyte structural changes, and ON biomechanical strains (explanted eyes) were evaluated. ROCK activity assays were conducted in separate ON regions. At 6W GL, mean RGC axon loss was 6.6 ± 13.3% in Rip and 36.3 ± 30.9% in BSS (P = 0.04, n = 10/group). RGC soma loss after crush was lower with Rip (68.6 ± 8.2%) than BSS (80.5 ± 5.7%, P = 0.006, n = 10/group). After 6W GL, RGC soma loss was lower with Rip (34 ± 5.0%) than BSS (51 ± 8.1%, P = 0.03, n = 10/group). Axonal transport of APP within the unmyelinated ON (UON) was unaffected by Rip. Maximum principal mechanical strains increased similarly in Rip and BSS-treated mice. Retinal ROCK 1 and 2 activity was reduced by Rip in GL eyes. The pROCK2/ROCK2 protein ratio rose in the retina of BSS GL eyes, but not in Rip GL eyes. Topical Rip reduced RGC loss in GL and ON crush, with suppression of ROCK signaling in the retina and ON. The neuroprotection mechanisms appear to involve effects on both RGC and astrocyte responses to IOP elevation.