Detection of esterified cholesterol in murine Bruch's membrane wholemounts with a perfringolysin O-based cholesterol marker

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.