Axonal Protection by Ripasudil, a Rho Kinase Inhibitor, via Modulating Autophagy in TNF-Induced Optic Nerve Degeneration
Axonal Protection by Ripasudil, a Rho Kinase Inhibitor, via Modulating Autophagy in TNF-Induced Optic Nerve Degeneration
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DOI：
10.1167/iovs.17-22000
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发表时间：
2017-10-01
影响因子：
4.4
通讯作者：
Takagi, Hitoshi
中科院分区：
文献类型：
--
作者：
Kitaoka, Yasushi;Sase, Kana;Takagi, Hitoshi
目的. Rho激酶抑制剂利帕舒地尔降低眼内压，但其在视神经轴突损伤中的作用尚待阐明。因此，我们研究了利帕舒地尔是否调节TNF诱导的轴突损失，并影响自噬机制诱导后的视神经变性。大鼠玻璃体腔注射肿瘤坏死因子，同时注射利帕舒地尔和肿瘤坏死因子或单独注射利帕舒地尔。计数轴突数目以评估利帕舒地尔对轴突损失的影响。免疫印迹分析检测p62和LC 3-II在视神经中的表达。电子显微镜用于确定轴突和神经胶质中的自噬体数量。进行免疫金标记以评估轴突中的自噬体。玻璃体内注射Ripasudil导致对TNF诱导的轴突损失的显著神经保护。玻璃体内注射TNF上调视神经中的p62，但利帕舒地尔完全抑制了这种增量。与基线相比，单独注射利帕舒地尔显著降低了p62和增强了LC 3-II蛋白水平。利帕舒地尔诱导的上调LC 3-II的TNF注射后，免疫组化分析显示，LC 3共定位在神经纤维。电镜分析显示，自噬体存在于轴突和胶质细胞中，尽管自噬体数量在利帕舒地尔注射后仅在轴突中显著增加。这些结果表明，利帕舒地尔增强轴突内自噬至少部分参与轴突保护。
PURPOSE. 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.METHODS. 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.RESULTS. 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.CONCLUSIONS. These results suggest that ripasudil-enhanced intra-axonal autophagy is at least partly involved in axonal protection.