glanatec and Optic-Nerve-Diseases

To elucidate molecular pharmacology of Rho-associated coiled-coil containing protein kinase inhibitors (ROCK-i, Ripasudil and Y27632) on their efficiency for aqueous outflow, 2D or 3D cultures of a human trabecular meshwork (HTM) were prepared in the presence of TGFβ2. Those were examined by transendothelial electrical resistance (TEER, 2D), electronic microscopy (EM, 2D and 3D), expression of the extracellular matrix (ECM) including collagen1 (COL1), COL4 and COL6, and fibronectin (FN) by immunolabeling and/or quantitative PCR (3D), and solidity of 3D organoids by a micro-squeezer. TGFβ2 significantly increased the TEER values in 2D cultures, and the ECM expression indicated that the 3D organoids assumed a more densely packed shape. ROCK-i greatly reduced the TGFβ2-induced enhancement of TEER and the immunolabeled ECM expression of the 3D organoids. In contrast, the mRNA expression of COL1 was increased, and those of COL4 and FN were unchanged. EM revealed that TGFβ2 caused the HTM cells to become more compact and abundant ECM deposits within the 3D organoids were observed. These were significantly inhibited by ROCK-i. The dense solids caused by the presence of TGFβ2 were significantly suppressed by ROCK-i. Current study indicates that ROCK-i cause beneficial effects toward the spatial configuration of TGFβ2-induced HTM 3D organoids.

Topics: Amides; Antihypertensive Agents; Cell Culture Techniques; Cell Line; Glaucoma, Open-Angle; Humans; Intraocular Pressure; Isoquinolines; Microscopy, Electron, Scanning; Optic Nerve Diseases; Organoids; Pyridines; rho-Associated Kinases; Spheroids, Cellular; Sulfonamides; Trabecular Meshwork; Transforming Growth Factor beta2

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.

Topics: Animals; Autophagy; Axons; Cell Count; Cytoprotection; Disease Models, Animal; Immunoblotting; Intravitreal Injections; Isoquinolines; Male; Microtubule-Associated Proteins; Nerve Degeneration; Neuroprotective Agents; Optic Nerve Diseases; Rats; Rats, Wistar; rho-Associated Kinases; Sequestosome-1 Protein; Sulfonamides; Tumor Necrosis Factor-alpha; Up-Regulation