Fasudil, a Clinically Used ROCK Inhibitor, Stabilizes Rod Photoreceptor Synapses after Retinal Detachment.
Fasudil, a Clinically Used ROCK Inhibitor, Stabilizes Rod Photoreceptor Synapses after Retinal Detachment.
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DOI：
10.1167/tvst.6.3.22
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发表时间：
2017-06
影响因子：
3
通讯作者：
Zarbin M
中科院分区：
文献类型：
--
作者：
Townes-Anderson E;Wang J;Halász É;Sugino I;Pitler A;Whitehead I;Zarbin M
视网膜脱离通过杆状轴突的回缩破坏外丛状层中的杆状双极突触。我们发现，断裂是由于 RhoA 激活所致，而使用 Y27632 抑制 Rho 激酶 (ROCK) 可减少突触损伤。我们测试用于其他临床应用的 ROCK 抑制剂法舒地尔是否可以预防脱离后的突触损伤。通过视网膜下注射平衡盐溶液 (BSS) 或法舒地尔 (1, 10 mM) 在猪中进行分离。在一些动物中，在 BSS 诱导的脱离后，将法舒地尔注射到玻璃体内。 2至4小时后，固定视网膜进行免疫细胞化学和共聚焦显微镜检查。通过外核层中突触小泡标记的成像来量化轴突回缩。使用碘化丙啶染色分析细胞凋亡。为了通过蛋白质印迹进行生化分析，将与视网膜色素上皮分离的视网膜外植体培养2小时。与对照脱离相比，视网膜下注射法舒地尔 (10 mM) 可使杆状小球的回缩减少 51.3%（n = 3 头猪，P = 0.002）。玻璃体内注射 10 mM 法舒地尔（一种临床上更可行的给药途径）也可减少回缩（28.7%，n = 5，P < 0.05）。对照组在 2 小时时没有光感受器变性，但到 4 小时时细胞凋亡就很明显。 Fasudil 10 mM 使核固缩减少 55.7% (n = 4, P < 0.001)。 30 μM 法舒地尔可降低 ROCK 下游效应子 cofilin 和肌球蛋白轻链的磷酸化（n = 8-10 个外植体，P < 0.05）。用法舒地尔抑制 ROCK 信号传导可减少视网膜脱离后光感受器变性并保留视杆双极突触。这些结果支持了先前用 Y27632 测试的可能性，即 ROCK 抑制可能减轻医源性脱离中的突触损伤。
Retinal detachment disrupts the rod-bipolar synapse in the outer plexiform layer by retraction of rod axons. We showed that breakage is due to RhoA activation whereas inhibition of Rho kinase (ROCK), using Y27632, reduces synaptic damage. We test whether the ROCK inhibitor fasudil, used for other clinical applications, can prevent synaptic injury after detachment. Detachments were made in pigs by subretinal injection of balanced salt solution (BSS) or fasudil (1, 10 mM). In some animals, fasudil was injected intravitreally after BSS-induced detachment. After 2 to 4 hours, retinae were fixed for immunocytochemistry and confocal microscopy. Axon retraction was quantified by imaging synaptic vesicle label in the outer nuclear layer. Apoptosis was analyzed using propidium iodide staining. For biochemical analysis by Western blotting, retinal explants, detached from retinal pigmented epithelium, were cultured for 2 hours. Subretinal injection of fasudil (10 mM) reduced retraction of rod spherules by 51.3% compared to control detachments (n = 3 pigs, P = 0.002). Intravitreal injection of 10 mM fasudil, a more clinically feasible route of administration, also reduced retraction (28.7%, n = 5, P < 0.05). Controls had no photoreceptor degeneration at 2 hours, but by 4 hours apoptosis was evident. Fasudil 10 mM reduced pyknotic nuclei by 55.7% (n = 4, P < 0.001). Phosphorylation of cofilin and myosin light chain, downstream effectors of ROCK, was decreased with 30 μM fasudil (n = 8–10 explants, P < 0.05). Inhibition of ROCK signaling with fasudil reduced photoreceptor degeneration and preserved the rod-bipolar synapse after retinal detachment. These results support the possibility, previously tested with Y27632, that ROCK inhibition may attenuate synaptic damage in iatrogenic detachments.