Rudolf Martin, Mohi Armin, Dettbarn Marie C, Miura Yoko, Aherrahrou Zouhair, Ranjbar Mahdy, Mutus Bulent, Knobloch Johannes K M
Department of Ophthalmology, University of Lübeck, Lübeck, Germany.
Institute for Integrative and Experimental Genomics, University of Lübeck, Lübeck, Germany.
Invest Ophthalmol Vis Sci. 2014 Jul 1;55(8):4759-67. doi: 10.1167/iovs.14-14311.
To investigate the effects of Bruch's membrane (BrM) neutral lipid deposition in mouse models and its significance to aging and age-related macular degeneration, it is essential to reliably detect small quantities of neutral lipids including esterified cholesterol (EC). In chorioretinal sections and BrM wholemounts, we tested a novel fluorescent cholesterol marker based on the bacterial toxin perfringolysin O (PFO) and compared results with those obtained with the classic cholesterol dye filipin.
An engineered plasmid containing the specific cholesterol binding domain (D4) of PFO fused to green fluorescent protein (GFP) was expressed in cultured E. coli, isolated, purified, and concentrated. A total of 150 BrM-choroid wholemounts and chorioretinal sections of 11- to 13-month-old ApoE(null) mice were prepared and stained with PFO/D4-GFP or filipin for EC. Samples were examined by epifluorescence microscopy.
The fluorescence intensity of PFO/D4-GFP was strong, stable, and, if small quantities of EC were present, superior to filipin. In all specimens, we could sharply locate the PFO/D4-GFP signal to BrM. A semiquantitative evaluation of BrM lipid deposition is possible by measuring PFO/D4-GFP fluorescence intensity.
The use of PFO/D4-GFP allowed a robust and direct detection of EC in aged murine BrM. In wholemount samples, its strong and stable fluorescence facilitated a semiquantitative evaluation of BrM-EC content over a large area. The patterns of EC deposition in murine BrM wholemounts are comparable with findings in human BrM wholemounts. Perfringolysin O/D4-GFP could be an important tool for investigating the effects of BrM lipid deposition in mouse models.
为了研究布鲁赫膜（BrM）中性脂质沉积在小鼠模型中的作用及其对衰老和年龄相关性黄斑变性的意义，可靠检测包括酯化胆固醇（EC）在内的少量中性脂质至关重要。在脉络膜视网膜切片和BrM整装片中，我们测试了一种基于产气荚膜梭菌溶血素O（PFO）的新型荧光胆固醇标记物，并将结果与经典胆固醇染料制霉菌素的结果进行比较。
将含有与绿色荧光蛋白（GFP）融合的PFO特异性胆固醇结合结构域（D4）的工程质粒在培养的大肠杆菌中表达，分离、纯化并浓缩。制备了150个11至13月龄载脂蛋白E基因敲除（ApoE(null)）小鼠的BrM -脉络膜整装片和脉络膜视网膜切片，并用PFO/D4 - GFP或制霉菌素对EC进行染色。通过落射荧光显微镜检查样本。
PFO/D4 - GFP的荧光强度强且稳定，并且在存在少量EC的情况下，优于制霉菌素。在所有标本中，我们可以将PFO/D4 - GFP信号精确地定位到BrM。通过测量PFO/D4 - GFP荧光强度，可以对BrM脂质沉积进行半定量评估。
使用PFO/D4 - GFP能够在衰老小鼠的BrM中进行强大而直接的EC检测。在整装片样本中，其强而稳定的荧光有助于对大面积的BrM - EC含量进行半定量评估。小鼠BrM整装片中EC沉积模式与人类BrM整装片中的发现相当。产气荚膜梭菌溶血素O/D4 - GFP可能是研究小鼠模型中BrM脂质沉积作用的重要工具。
