April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Detection of Esterified Cholesterol in Murine Bruch’s Membrane Wholemounts with a Novel Cholesterol Dye
Author Affiliations & Notes
  • M. C. Dettbarn
    Department of Ophthalmology,
    University Medical Center Schleswig-Holstein, Campus Luebeck, Germany, Luebeck, Germany
  • J. Knobloch
    Institute for Microbiology and Hygiene,
    University Medical Center Schleswig-Holstein, Campus Luebeck, Germany, Luebeck, Germany
  • S. Grisanti
    Department of Ophthalmology,
    University Medical Center Schleswig-Holstein, Campus Luebeck, Germany, Luebeck, Germany
  • M. Rudolf
    Department of Ophthalmology,
    University Medical Center Schleswig-Holstein, Campus Luebeck, Germany, Luebeck, Germany
  • Footnotes
    Commercial Relationships  M.C. Dettbarn, None; J. Knobloch, None; S. Grisanti, None; M. Rudolf, None.
  • Footnotes
    Support  International Retinal Research Foundation, USA; Jackstaedt - Stiftung, Germany;
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 4937. doi:
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    • Get Citation

      M. C. Dettbarn, J. Knobloch, S. Grisanti, M. Rudolf; Detection of Esterified Cholesterol in Murine Bruch’s Membrane Wholemounts with a Novel Cholesterol Dye. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4937.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

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 reliable detect lipids including esterified cholesterol (EC). In murine BrM wholemounts we tested the novel fluorescent cholesterol dye GFP-D4 derived from the bacterial toxin Perfringolysin O (PFO) and compared results to those obtained with 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. GFP-D4 was isolated, purified, and concentrated. Ten BrM-choroid wholemounts of 8-10-month-old ApoEnull mice were 1) prepared with clean removal of RPE and retina, 2) stained with GFP-D4 (80 µg/ml) for EC following ethanol extraction and cholesterol esterase treatment and 3) examined by epifluorescence microscopy.

Results: : The fluorescence intensity of freshly isolated GFP-D4 was strong, stable, and superior to filipin. In all specimens we could sharply locate the GFP-D4 signal to BrM. Across a wholemount sample we found a laminar pattern of multiple defined dots representing EC in lipid vesicles present in aged murine BrM. A semi-quantitative evaluation of BrM lipid deposition is possible by measuring GFP-D4 fluorescence intensity.

Conclusions: : GFP-D4 allowed a robust and direct detection of EC in aged murine BrM. In wholemount samples its strong and stable fluorescence facilitated a semi-quantitative evaluation of BrM’s EC content over a large area. The patterns of EC deposition in murine BrM wholemounts are comparable to findings in human BrM wholemounts (Rudolf et al. 2009). GFP-D4 could be an important tool for investigating the effects of BrM lipid deposition in mouse models.

Keywords: Bruch's membrane • lipids • age-related macular degeneration 
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