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Kent Y. Feng, Jindong Ding, Una Kelly, Catherine Bowes Rickman; Age-dependent Distribution of Glycosaminoglycans in Mouse Bruch’s Membrane. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2327.
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Work from our group has demonstrated that heparan sulfate (HS) in the Bruch’s membrane (BrM) and choroid can inhibit the alternative pathway of complement (APC). Dysregulation of the complement cascade, specifically the APC, is a critical predisposing step in age-related macular degeneration (AMD) development. We hypothesized that with age and disease there is a change in the concentration, distribution and chemical composition of the HS in BrM and that this could affect the degree of complement activation at this site.
Murine eyes were fixed in 2% paraformaldehyde/2% glutaraldehyde and embedded in agar. Vibratome sections of 100 µm were cut and stained with Cupromeronic Blue, which selectively stains sulfated glycoaminoglycans (GAGs). Selected control sections were digested with heparinase III and chondroitinase. Stained sections were embedded in Epon-Spurr’s resin, cut into 80 nm sections, and stained with uranyl acetate. Transmission electron microscopy images of the BrM were acquired using a FEI Technai TEM.
The general density of sulfated GAGs increased with mouse age, evidenced by increased number of stained elements. Particularly in mice aged over 65 weeks, there was an increase in thick filamentous staining in the basal lamina of the RPE that is susceptible to heparinase digestion. Additionally, much of the staining in the RPE basal lamina and inner and outer collageous layers (ICL/OCL) of BrM were absent in heparinase-treated tissues compared to non-treated tissues. Following chondroitinase treatment Cupromeronic Blue staining was decreased in the areas closer to the central elastin layer whereas similar levels of staining was observed in the ICL and OCL compared to non-treated tissues.
Overall levels of sulfated GAGs appear to increase with age. The most notable change was the formation of the type 5 HS filaments (described by Call and Hollyfield) in aged mice. Our previous studies showing HS inhibits the APC and these observed changes in sulfated GAGs in BrM support our hypothesis and suggest that this could contribute to the pathogenesis of AMD.
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