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

PURPOSE: 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.

METHODS: 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.

RESULTS: 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.

CONCLUSIONS: 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.