title: Detection of esterified cholesterol in murine Bruch's membrane wholemounts with a perfringolysin O-based cholesterol marker.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Ophthalmology, University of Lübeck, Lübeck, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Rudolf
authors.affiliation: Department of Ophthalmology, University of Lübeck, Lübeck, Germany.
authors.forename: Armin
authors.initials: A
authors.lastname: Mohi
authors.affiliation: Department of Ophthalmology, University of Lübeck, Lübeck, Germany.
authors.forename: Marie C
authors.initials: MC
authors.lastname: Dettbarn
authors.affiliation: Department of Ophthalmology, University of Lübeck, Lübeck, Germany.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Miura
authors.affiliation: Institute for Integrative and Experimental Genomics, University of Lübeck, Lübeck, Germany.
authors.forename: Zouhair
authors.initials: Z
authors.lastname: Aherrahrou
authors.affiliation: Department of Ophthalmology, University of Lübeck, Lübeck, Germany.
authors.forename: Mahdy
authors.initials: M
authors.lastname: Ranjbar
authors.affiliation: Department of Chemistry & Biochemistry, University of Windsor, Windsor, Ontario, Canada.
authors.forename: Bulent
authors.initials: B
authors.lastname: Mutus
authors.affiliation: Institute for Medical Microbiology & Hygiene, University of Lübeck, Lübeck, Germany.
authors.forename: Johannes K M
authors.initials: JK
authors.lastname: Knobloch
mesh_terms: D000375:Aging / Q000378:metabolism* / Q000473:pathology; D000818:Animals; D001427:Bacterial Toxins; D016570:Bruch Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured; D002788:Cholesterol Esters / Q000378:metabolism*; D003016:Clostridium perfringens; D004195:Disease Models, Animal; D005240:Feasibility Studies; D005260:Female; D006460:Hemolysin Proteins; D006801:Humans; D008268:Macular Degeneration / Q000175:diagnosis* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D046529:Microscopy, Electron, Transmission
chemical_list: D001427:Bacterial Toxins; D002788:Cholesterol Esters; D006460:Hemolysin Proteins; C020452:Clostridium perfringens theta-toxin
keywords: Bruch's membrane; aging; cholesterol marker; lipids; mouse model