Yang Jialiang, Wu Haiping, Li Qi, Guo Jiaxin, Yao Jialin, Pan Shengliu, Wu Xiawei, Huang Haotian, Chen Runfang, Chen Junnian, Wang Yubin, Peng Yue, Wu Fuhua, Hu Jing
Sichuan Provincial Key Laboratory for Human Disease Gene Study, Department of Laboratory Medicines, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu, China.
Department of Clinical Pharmacology, College of Pharmacy, Dalian Medical University, Dalian, China.
Transl Vis Sci Technol. 2025 May 1;14(5):14. doi: 10.1167/tvst.14.5.14.
Retina and retinal pigment epithelium (RPE) wholemounts are important models for studying the pathophysiology of retinal-related ophthalmic diseases, such as age-related macular degeneration and diabetic retinopathy. Currently, there is no method available for simultaneously obtaining retina and RPE wholemounts. The aim of this study is to develop a simple, rapid, and effective technique for the simultaneous isolation of mouse retina and RPE wholemounts.
We developed a novel, streamlined procedure for the efficient isolation of intact retina and RPE wholemounts from mouse eyes. The method involves minimal dissection and uses basic laboratory equipment, allowing the entire process to be completed in approximately 2 to 5 minutes per sample. The study also explores the impact of different fixation times on the structural integrity and quality of both retina and RPE wholemounts (3 hours in 4% paraformaldehyde [PFA], 30 minutes < 1 × phosphate-buffered saline [PBS] < 3 hours).
The new method consistently yields high-quality, intact retina and RPE wholemounts, with excellent structural integrity suitable for downstream imaging and molecular analyses. The technique significantly reduces preparation time. Optimal fixation conditions were identified, with 3 hours of fixation in 4% PFA and PBS incubation times between 30 minutes and 3 hours yielding the best results. The approach showed higher tissue integrity (80% vs. 45%) and improved staining quality of photoreceptor and ganglion cells. Additionally, the method is highly reproducible and effective for wholemount preparations from both young and older mice (6 and 12 months).
This study presents a significant advancement in the preparation of retina and RPE wholemounts. The simplicity, speed, and preservation of tissue integrity of the new method make it a valuable tool for ophthalmic disease research. Its potential applications include drug screening, gene therapy, and disease modeling, offering significant advantages in time efficiency, reproducibility, and the quality of morphological analysis.
视网膜和视网膜色素上皮（RPE）整装标本是研究视网膜相关眼科疾病（如年龄相关性黄斑变性和糖尿病视网膜病变）病理生理学的重要模型。目前，尚无同时获取视网膜和RPE整装标本的方法。本研究的目的是开发一种简单、快速且有效的技术，用于同时分离小鼠视网膜和RPE整装标本。
我们开发了一种新颖、简化的程序，用于从小鼠眼中高效分离完整的视网膜和RPE整装标本。该方法所需解剖操作极少，并使用基本的实验室设备，每个样本的整个过程大约可在2至5分钟内完成。本研究还探讨了不同固定时间对视网膜和RPE整装标本结构完整性和质量的影响（在4%多聚甲醛[PFA]中固定3小时，在1×磷酸盐缓冲盐水[PBS]中处理30分钟＜＜3小时）。
新方法始终能产生高质量、完整的视网膜和RPE整装标本，具有出色的结构完整性，适用于下游成像和分子分析。该技术显著缩短了制备时间。确定了最佳固定条件，在4% PFA中固定3小时，PBS孵育时间在30分钟至3小时之间可获得最佳结果。该方法显示出更高的组织完整性（80%对45%），并改善了感光细胞和神经节细胞的染色质量。此外，该方法对于年轻和老年小鼠（6个月和12个月）的整装标本制备具有高度可重复性且有效。
本研究在视网膜和RPE整装标本制备方面取得了重大进展。新方法的简单性、速度和组织完整性的保持使其成为眼科疾病研究的有价值工具。其潜在应用包括药物筛选、基因治疗和疾病建模，在时间效率、可重复性和形态学分析质量方面具有显著优势。
