Axonal Protection by Ripasudil, a Rho Kinase Inhibitor, via Modulating Autophagy in TNF-Induced Optic Nerve Degeneration

• Published in: Investigative ophthalmology & visual science Vol. 58; no. 12; pp. 5056 - 5064
• Main Authors: Kitaoka, Yasushi, Sase, Kana, Tsukahara, Chihiro, Kojima, Kaori, Shiono, Akira, Kogo, Jiro, Tokuda, Naoto, Takagi, Hitoshi
• Format: Journal Article
• Language: English
• Published: United States 01-10-2017
• Subjects: Animals; Autophagy - drug effects; Axons - drug effects; Axons - pathology; Cell Count; Cytoprotection; Disease Models, Animal; Immunoblotting; Intravitreal Injections; Isoquinolines - pharmacology; Male; Microtubule-Associated Proteins - metabolism; Nerve Degeneration - metabolism; Nerve Degeneration - pathology; Nerve Degeneration - prevention & control; Neuroprotective Agents - pharmacology; Optic Nerve Diseases - metabolism; Optic Nerve Diseases - pathology; Optic Nerve Diseases - prevention & control; Rats; Rats, Wistar; rho-Associated Kinases - antagonists & inhibitors; Sequestosome-1 Protein - metabolism; Sulfonamides - pharmacology; Tumor Necrosis Factor-alpha - toxicity; Up-Regulation
• ISSN: 1552-5783; 1552-5783
• DOI: 10.1167/iovs.17-22000

The Rho kinase inhibitor ripasudil decreases intraocular pressure, although its role in optic nerve axonal damage should be clarified. We therefore investigated whether ripasudil modulates TNF-induced axonal loss and affects autophagy machinery after the induction of optic nerve degeneration.
Rats were given intravitreal injection of TNF, concomitant injection of ripasudil hydrochloride hydrate and TNF, or ripasudil alone. Axon numbers were counted to evaluate the effects of ripasudil against axon loss. Immunoblot analysis was performed to examine p62 as well as LC3-II expression in optic nerves. Electron microscopy was used to determine autophagosome numbers in axons and glia. Immunogold labeling was performed to evaluate autophagosomes in axons.
Ripasudil injected intravitreally resulted in significant neuroprotection against TNF-induced axon loss. Intravitreal TNF injection upregulated p62 in the optic nerve, but ripasudil completely inhibited this increment. The ripasudil alone injection diminished p62 and enhanced LC3-II protein levels significantly compared with baseline. Ripasudil-induced upregulation of LC3-II was seen after TNF injection, and immunohistochemical analysis revealed that LC3 colocalized in nerve fibers. Electron microscopic analysis revealed that autophagosomes were present in axons and glia, although autophagosome numbers increased significantly after ripasudil injection only in axons.
These results suggest that ripasudil-enhanced intra-axonal autophagy is at least partly involved in axonal protection.