Shaw Peter X, Sang Alan, Wang Yan, Ho Daisy, Douglas Christopher, Dia Lara, Goldberg Jeffrey L
Department of Ophthalmology and Shiley Eye Institute, University of California San Diego, La Jolla, CA 92093, United States.
Department of Ophthalmology and Shiley Eye Institute, University of California San Diego, La Jolla, CA 92093, United States; Byers Eye Institute, Stanford University, Palo Alto, CA 94303, United States.
Exp Eye Res. 2017 May;158:33-42. doi: 10.1016/j.exer.2016.07.006. Epub 2016 Jul 18.
Intraocular pressure (IOP)-lowering ophthalmic solutions that inhibit Rho-associated protein kinases (Rock) and norepinephrine transporters (Net) are currently under clinical evaluation. Here we evaluate topical application of one such drug for its effects on retinal ganglion cell (RGC) survival and axon regeneration after optic nerve crush injury. We performed unilateral optic nerve crush on young rats (P18) and topically applied Rock/Net inhibitor AR-13324 or placebo 3 times a day for 14 days. IOP was measured starting 3 days before and up to 9 days after injury. On day 12, cholera toxin B (CTB) was injected intravitreally to trace optic nerve regeneration. On day 14, retinas and optic nerves were collected. The retinas were flat-mounted and stained with RBPMS to quantify RGC survival and the optic nerves were sectioned for optic nerve axon quantification using fluorescent and confocal microscopy. Rock phosphorylation targets implicated in axon growth including cofilin and LIMK were examined by fluorescence microscopy and quantitative western blotting. AR-13324 lowered IOP as expected. RGC survival and optic nerve axon regeneration were significantly higher with Rock/Net inhibitor treatment compared with placebo. Furthermore, topical therapy decreased Rock target protein phosphorylation in the retinas and proximal optic nerves. These data suggest that topical administration of a Rock/Net inhibitor promotes RGC survival and regeneration after optic nerve injury, with associated molecular changes indicative of posterior drug activity. Coordinated IOP lowering and neuroprotective or regenerative effects may be advantageous in the treatment of patients with glaucoma.
目前，抑制Rho相关蛋白激酶（Rock）和去甲肾上腺素转运体（Net）的降眼压眼科溶液正在进行临床评估。在此，我们评估了一种此类药物局部应用对视神经挤压损伤后视网膜神经节细胞（RGC）存活和轴突再生的影响。我们对幼鼠（P18）进行单侧视神经挤压，并每天局部应用Rock/Net抑制剂AR - 13324或安慰剂3次，持续14天。在损伤前3天开始直至损伤后9天测量眼压。在第12天，玻璃体内注射霍乱毒素B（CTB）以追踪视神经再生。在第14天，收集视网膜和视神经。将视网膜平铺固定并用RBPMS染色以量化RGC存活情况，将视神经切片，使用荧光和共聚焦显微镜对视神经轴突进行量化。通过荧光显微镜和定量蛋白质免疫印迹法检测与轴突生长相关的Rock磷酸化靶点，包括丝切蛋白和LIMK。正如预期的那样，AR - 13324降低了眼压。与安慰剂相比，Rock/Net抑制剂治疗组的RGC存活和视神经轴突再生显著更高。此外，局部治疗降低了视网膜和近端视神经中Rock靶点蛋白的磷酸化水平。这些数据表明，局部应用Rock/Net抑制剂可促进视神经损伤后RGC的存活和再生，相关的分子变化表明药物具有后向活性。协调降眼压以及神经保护或再生作用可能对青光眼患者的治疗有利。
