ABSTRACT

Retinal ischemia is a major cause of vision loss and a common underlying mechanism associated with diseases, such as diabetic retinopathy and central retinal artery occlusion. We have previously demonstrated the robust neuroprotection in retina induced by post-conditioning (post-C), a brief period of ischemia, 24 h, following a prolonged and damaging initial ischemia. The mechanisms underlying post-C-mediated retinal protection are largely uncharacterized. We hypothesized that macroautophagy/autophagy is a mediator of post-C-induced neuroprotection. This study employed an in vitro model of oxygen glucose deprivation (OGD) in the retinal R28 neuronal cell line, and an in vivo rat model of retinal ischemic injury. In vivo, there were significant increases in autophagy proteins, MAP1LC3-II/LC3-II, and decreases in SQSTM1/p62 (sequestosome 1) in ischemia/post-C vs. ischemia/sham post-C. Blockade of Atg5 and Atg7 in vivo decreased LC3-II, increased SQSTM1, attenuated the functional protective effect of post-C, and increased histological damage and TUNEL compared to non-silencing siRNA. TUNEL after ischemia in vivo was found in retinal ganglion, amacrine, and photoreceptor cells. Blockade of Atg5 attenuated the post-C neuroprotection by a brief period of OGD in vitro. Moreover, in vitro, post-C attenuated cell death, loss of cellular proliferation, and defective autophagic flux from prolonged OGD. Stimulating autophagy using Tat-Beclin 1 rescued retinal neurons from cell death after OGD. As a whole, our results suggest that autophagy is required for the neuroprotective effect of retinal ischemic post-conditioning and augmentation of autophagy offers promise in the treatment of retinal ischemic injury.

Abbreviations: BECN1: Beclin 1, autophagy related; DAPI: 4’,6-diamidino-2-phenylindole; DR: diabetic retinopathy; EdU: 5-ethynyl-2’-deoxyuridine; ERG: Electroretinogram; FITC: Fluorescein isothiocyanate; GCL: Ganglion cell layer; GFAP: Glial fibrillary acidic protein; INL: Inner nuclear layer; IPL: Inner plexiform layer; MAP1LC3/LC3: Microtubule-associated protein 1 light chain 3; OGD: Oxygen-glucose deprivation; ONL: Outer nuclear layer; OP: Oscillatory potential; PFA: Paraformaldehyde; PL: Photoreceptor layer; post-C: post-conditioning; RFP: Red fluorescent protein; RGC: Retinal ganglion cell; RPE: Retinal pigment epithelium; RT-PCR: Real-time polymerase chain reaction; SEM: Standard error of the mean; siRNA: Small interfering RNA; SQSTM1: Sequestosome 1; STR: Scotopic threshold response; Tat: Trans-activator of transcription; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling

摘要

视网膜缺血是视力丧失的主要原因，也是与糖尿病性视网膜病变和视网膜中央动脉阻塞等疾病相关的常见潜在机制。我们之前已经证明了后处理（post-C），短暂的缺血时间，24 小时，在长期和破坏性的初始缺血后诱导的视网膜中强大的神经保护作用。后 C 介导的视网膜保护的机制在很大程度上是未知的。我们假设巨自噬/自噬是 C 后诱导的神经保护的介质。本研究中采用的体外氧葡萄糖剥夺（OGD）中的视网膜R28神经元细胞系的模型，以及在体内视网膜的缺血性损伤的大鼠模型。体内，自噬蛋白 MAP1LC3-II/LC3-II 显着增加，缺血/C 后与. C 后缺血/假手术。的封锁的Atg5和ATG7体内降低LC3-II，增加SQSTM1，减毒后-C的功能的保护作用，并且比非沉默的siRNA增加组织学损害和TUNEL。在体内局部缺血后，在视网膜神经节、无长突和感光细胞中发现了TUNEL 。Atg5 的阻断减弱了体外OGD 的短暂时期的 C 后神经保护作用。此外，体外, post-C 减弱了细胞死亡、细胞增殖的丧失和长期 OGD 导致的自噬流缺陷。使用 Tat-Beclin 1 刺激自噬可在 OGD 后从细胞死亡中拯救视网膜神经元。总的来说，我们的结果表明，自噬是视网膜缺血后处理的神经保护作用所必需的，而自噬的增强为治疗视网膜缺血性损伤提供了希望。

缩写：BECN1：Beclin 1，自噬相关；DAPI：4',6-二脒基-2-苯基吲哚；DR：糖尿病视网膜病变；EdU：5-乙炔基-2'-脱氧尿苷；ERG：视网膜电图；FITC：异硫氰酸荧光素；GCL：神经节细胞层；GFAP：胶质纤维酸性蛋白；INL：内核层；IPL：内丛状层；MAP1LC3/LC3：微管相关蛋白 1 轻链 3；OGD：氧糖剥夺；ONL：外核层；OP：振荡电位；PFA：多聚甲醛；PL：感光层；post-C：后处理；RFP：红色荧光蛋白；RGC：视网膜神经节细胞；RPE：视网膜色素上皮；RT-PCR：实时聚合酶链反应；SEM：平均值的标准误差；siRNA：小干扰RNA；SQSTM1：螯合体 1；STR：暗视阈值反应；Tat：转录的反式激活因子；隧道：