Wagner Natalie, Tsai Teresa, Reinehr Sabrina, Theile Janine, Dick H Burkhard, Joachim Stephanie C
Experimental Eye Research Institute, University Eye Hospital, Ruhr-University Bochum, Bochum, Germany.
Front Neurosci. 2024 Jul 24;18:1401571. doi: 10.3389/fnins.2024.1401571. eCollection 2024.
One of the most common causes of vision loss in the elderly population worldwide is age-related macular degeneration (AMD). Subsequently, the number of people affected by AMD is estimated to reach approximately 288 million by the year 2040. The aim of this study was to develop an model that simulates various aspects of the complex AMD pathogenesis.
For this purpose, primary porcine retinal pigment epithelial cells (ppRPE) were isolated and cultured. One group was exposed to medium containing sodium iodate (NaIO) to induce degeneration. The others were exposed to different supplemented media, such as bovine serum albumin (BSA), homogenized porcine retinas (HPR), or rod outer segments (ROOS) for eight days to promote retinal deposits. Then, these ppRPE cells were cocultured with porcine neuroretina explants for another eight days. To assess the viability of ppRPE cells, live/dead assay was performed at the end of the study. The positive RPE65 and ZO1 area was evaluated by immunocytochemistry and the expression of , , and was analyzed by RT-qPCR. Additionally, drusen (), inflammation (, , , , ), oxidative stress (, , ), and hypoxia () markers were investigated. The concentration of the inflammatory cytokines IL-6 and IL-8 was determined in medium supernatants from day 16 and 24 via ELISA.
Live/dead assay suggests that especially exposure to NaIO and HPR induced damage to ppRPE cells, leading in a significant ppRPE cell loss. All supplemented media resulted in decreased RPE-characteristic markers (RPE65; ZO-1) and gene expression like and in the cultured ppRPE cells. Besides, some inflammatory, oxidative as well as hypoxic stress markers were altered in ppRPE cells cultivated with NaIO. The application of HPR induced an enhanced expression. Pre-exposure of the ppRPE cells led to a diminished number of cones in all supplemented media groups compared to controls.
Overall, this novel coculture model represents an interesting initial approach to incorporating deposits into coculture to mimic AMD pathogenesis. Nevertheless, the effects of the media used need to be investigated in further studies.
全球老年人群视力丧失的最常见原因之一是年龄相关性黄斑变性（AMD）。据估计，到2040年，受AMD影响的人数将达到约2.88亿。本研究的目的是建立一个模拟复杂AMD发病机制各个方面的模型。
为此，分离并培养原代猪视网膜色素上皮细胞（ppRPE）。一组暴露于含碘酸钠（NaIO）的培养基中以诱导变性。其他组暴露于不同的补充培养基中，如牛血清白蛋白（BSA）、猪视网膜匀浆（HPR）或视杆细胞外段（ROOS），持续八天以促进视网膜沉积物形成。然后，将这些ppRPE细胞与猪神经视网膜外植体共培养另外八天。为评估ppRPE细胞的活力，在研究结束时进行活/死检测。通过免疫细胞化学评估阳性RPE65和ZO1区域，并通过RT-qPCR分析 、 和 的表达。此外，研究了玻璃膜疣（ ）、炎症（ 、 、 、 、 ）、氧化应激（ 、 ）和缺氧（ ）标志物。通过ELISA在第16天和第24天测定培养基上清液中炎症细胞因子IL-6和IL-8的浓度。
活/死检测表明，特别是暴露于NaIO和HPR会诱导ppRPE细胞损伤，导致ppRPE细胞显著损失。所有补充培养基均导致培养的ppRPE细胞中RPE特征性标志物（RPE65；ZO-1）和基因表达如 和 降低。此外，在用NaIO培养的ppRPE细胞中，一些炎症、氧化以及缺氧应激标志物发生了改变。HPR的应用诱导 表达增强。与对照组相比，ppRPE细胞的预暴露导致所有补充培养基组中的视锥细胞数量减少。
总体而言，这种新型共培养模型是将沉积物纳入共培养以模拟AMD发病机制的一种有趣的初步方法。然而，所用培养基的效果需要在进一步研究中进行调查。
