Wagner Natalie, Reinehr Sabrina, Gammel Maurice R, Greulich Andrea, Hurst José, Dick H Burkhard, Schnichels Sven, Joachim Stephanie C
Experimental Eye Research Institute, University Eye Hospital, Ruhr-University Bochum, Bochum, Germany.
University Eye Hospital, Centre for Ophthalmology, Tübingen, Germany.
Front Neurosci. 2020 Oct 6;14:556700. doi: 10.3389/fnins.2020.556700. eCollection 2020.
Age-related macular degeneration (AMD) is the leading cause of blindness in industrialized countries among people over 60 years. It has multiple triggers and risk factors, but despite intense research efforts, its pathomechanisms are currently not completely understood. AMD pathogenesis is characterized by soft drusen in Bruch's membrane and involves the retinal pigment epithelium-Bruch's membrane-choroid complex and adjacent structures, like photoreceptors. This study explores the potential of novel cultivation techniques to preserve photoreceptors in retinal explants to gain better insights in AMD pathology. The porcine retina explants were cultured for 4 and 8 days using three different explantation techniques, namely, control (photoreceptors facing down, touching the filter), filter (photoreceptors facing up, turned sample using a filter), and tweezers (photoreceptors facing up, turned sample using tweezers). Optical coherence tomography revealed that the tweezers method had the best capacity to limit thinning of the retinal explants. Both novel methods displayed advantages in maintaining outer segment thickness. Additionally, immunofluorescence evaluation revealed a better preservation of opsin cells and rhodopsin signal intensity in both novel methods, especially the tweezers method. Furthermore, RT-qPCR analysis demonstrated an upregulation of and mRNA expression in tweezers samples at 8 days. Amacrine and bipolar cell numbers were not altered at day 4 of cultivation, while cultivation until 8 days led to reduced bipolar cell numbers. At 4 days, mRNA was upregulated in filter samples, but expression was downregulated. Retinal ganglion cells were diminished in both novel techniques due to a direct physical contact with the insert. Remarkably, no difference in mRNA expression was detected among the techniques. Nevertheless, both novel methods exhibited an improved retention of photoreceptor cells. In conclusion, the tweezers technique was the most promising one. Due to the high homology of the porcine to the human retina, it provides a reasonable alternative to rodent models. Consequently, an adapted coculture system based on the current findings may serve as an model suitable to analyze AMD pathomechanisms and novel therapeutic approaches.
年龄相关性黄斑变性（AMD）是工业化国家60岁以上人群失明的主要原因。它有多种触发因素和风险因素，但尽管进行了大量研究，其发病机制目前尚未完全明确。AMD的发病机制以Bruch膜中的软性玻璃疣为特征，涉及视网膜色素上皮-Bruch膜-脉络膜复合体及相邻结构，如光感受器。本研究探索了新型培养技术在视网膜外植体中保存光感受器的潜力，以便更好地了解AMD病理学。使用三种不同的取材技术将猪视网膜外植体培养4天和8天，即对照（光感受器朝下，接触滤膜）、滤膜法（光感受器朝上，使用滤膜翻转样本）和镊子法（光感受器朝上，使用镊子翻转样本）。光学相干断层扫描显示，镊子法在限制视网膜外植体变薄方面能力最佳。两种新方法在维持外段厚度方面均显示出优势。此外，免疫荧光评估显示，两种新方法，尤其是镊子法，对视蛋白细胞和视紫红质信号强度的保存更好。此外，RT-qPCR分析表明，镊子法样本在8天时 和 mRNA表达上调。培养4天时，无长突细胞和双极细胞数量未改变，但培养至8天时双极细胞数量减少。在4天时，滤膜法样本中 mRNA上调，但 表达下调。由于与插入物直接物理接触，两种新技术中的视网膜神经节细胞均减少。值得注意的是，各技术之间未检测到 mRNA表达的差异。然而，两种新方法均显示出光感受器细胞保留率提高。总之，镊子技术是最有前景的技术。由于猪视网膜与人视网膜具有高度同源性，它为啮齿动物模型提供了合理的替代方案。因此，基于当前研究结果的适应性共培养系统可作为一个适合分析AMD发病机制和新型治疗方法的模型。
