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