Su Erin, Kesavamoorthy Niranjana, Junge Jason A, Zheng Mengmei, Craft Cheryl Mae, Ameri Hossein
Department of Ophthalmology, USC Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
Department of Biological Sciences, David Dornsife College of Letters Arts and Sciences, University of Southern California Dana, Los Angeles, CA 90089, USA.
Curr Issues Mol Biol. 2024 Jan 6;46(1):612-620. doi: 10.3390/cimb46010039.
Fluorescence lifetime imaging microscopy (FLIM) is a technique that analyzes the metabolic state of tissues based on the spatial distribution of fluorescence lifetimes of certain interacting molecules. We used multiphoton FLIM to study the metabolic state of developing C57BL6/J and rd10 retinas based on the fluorescence lifetimes of free versus bound nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD(P)H), with free NAD(P)H percentages suggesting increased glycolysis and bound NAD(P)H percentages indicating oxidative phosphorylation. The mice were sacrificed and enucleated at various time points throughout their first 3 months of life. The isolated eyecups were fixed, sectioned using a polyacrylamide gel embedding technique, and then analyzed with FLIM. The results suggested that in both C57BL6/J mice and rd10 mice, oxidative phosphorylation initially decreased and then increased, plateauing over time. This trend, however, was accelerated in rd10 mice, with its turning point occurring at p10 versus the p30 turning point in C57BL6/J mice. There was also a noticeable difference in oxidative phosphorylation rates between the outer and inner retinas in both strains, with greater oxidative phosphorylation present in the latter. A greater understanding of rd10 and WT metabolic changes during retinal development may provide deeper insights into retinal degeneration and facilitate the development of future treatments.
荧光寿命成像显微镜（FLIM）是一种基于某些相互作用分子荧光寿命的空间分布来分析组织代谢状态的技术。我们使用多光子FLIM，基于游离与结合的烟酰胺腺嘌呤二核苷酸和烟酰胺腺嘌呤二核苷酸磷酸（NAD(P)H）的荧光寿命，研究发育中的C57BL6/J和rd10视网膜的代谢状态，游离NAD(P)H百分比表明糖酵解增加，结合NAD(P)H百分比表明氧化磷酸化。在小鼠出生后的前3个月内的不同时间点将其处死并摘除眼球。分离出的眼杯经固定后，采用聚丙烯酰胺凝胶包埋技术切片，然后用FLIM进行分析。结果表明，在C57BL6/J小鼠和rd10小鼠中，氧化磷酸化最初下降，然后上升，随时间趋于平稳。然而，这种趋势在rd10小鼠中加速，其转折点出现在出生后第10天，而C57BL6/J小鼠的转折点出现在出生后第30天。在这两个品系的小鼠中，视网膜外层和内层的氧化磷酸化速率也存在明显差异，后者的氧化磷酸化程度更高。更深入地了解rd10和野生型小鼠视网膜发育过程中的代谢变化，可能会为视网膜变性提供更深入的见解，并促进未来治疗方法的开发。
