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

TY - JOUR

T1 - Detection of esterified cholesterol in murine Bruch's membrane wholemounts with a perfringolysin O-based cholesterol marker

AU - Rudolf, Martin

AU - Mohi, Armin

AU - Dettbarn, Marie C

AU - Miura, Yoko

AU - Aherrahrou, Zouhair

AU - Ranjbar, Mahdy

AU - Mutus, Bulent

AU - Knobloch, Johannes K-M

N1 - Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

PY - 2014/7/1

Y1 - 2014/7/1

N2 - 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.

AB - 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.

KW - Aging

KW - Animals

KW - Bacterial Toxins

KW - Bruch Membrane

KW - Cells, Cultured

KW - Cholesterol Esters

KW - Clostridium perfringens

KW - Disease Models, Animal

KW - Feasibility Studies

KW - Female

KW - Hemolysin Proteins

KW - Humans

KW - Macular Degeneration

KW - Mice

KW - Mice, Inbred C57BL

KW - Microscopy, Electron, Transmission

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1167/iovs.14-14311

DO - 10.1167/iovs.14-14311

M3 - SCORING: Journal article

C2 - 24985479

VL - 55

SP - 4759

EP - 4767

JO - INVEST OPHTH VIS SCI

JF - INVEST OPHTH VIS SCI

SN - 0146-0404

IS - 8

ER -