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C-M Li, K Bradley, BH Chung, CL Millican, CA Curcio; Absence of Bruch’s Membrane (BrM) Cholesterol Deposition in Rabbits Consuming Atherogenic Diets . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2813.
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Purpose: To determine the effect of diet-induced hypercholesterolemia on BrM, choroid, and optic nerve of rabbits. Methods: 1) Female New Zealand white rabbits (8± 0.9 lb) were maintained on normal chow (n=6), normal chow with 2% cholesterol (Diet1, n=8, 97-190 days), and a semi-purified diet (Diet2, 95-190 days). 2) Concentrations of cholesterol in plasma very-low, low-, and high-density lipoprotein fractions (VLDL, LDL, HDL) and total cholesterol (TC) were determined by semi-automated density gradient separation and enzymatic colorimetric assay at baseline and at sacrifice. 3) Eyes were preserved by immersion (n=16) or transcardiac perfusion (n=8) with 2.5% glutaraldehyde and 1% paraformaldehyde. Eyes were processed for electron microscopic evaluation of lipid particles and light microscopic lipid histochemistry. Filipin was used to demonstrate unesterified cholesterol (UC) and esterified cholesterol (EC, following extraction and hydrolysis). Oil red O was used to demonstrate neutral lipids (EC and triglycerides). 4) Lipids were extracted with chloroform and methanol from optic nerves of 2 control and 2 Diet1 animals for analysis of EC by mass spectrometry. Lipids were separated by reversed phase HPLC using an isopropanol gradient in 10 mM ammonium acetate. Positive ion mass spectra of ammonium ion adducts of cholesteryl esters were compared to standards. Results: 1) Both diets produced significantly higher TC than controls, with the greatest increases in the VLDL and LDL fractions. Diet1 had significantly greater LDL and lower HDL cholesterol than Diet2. 2) Choroids of Diet1 and Diet2 animals contained cholesterol crystals and macrophages with cholesterol-rich droplets. Macrophages formed a discrete layer in the outer choroid that was thickest in Diet1 animals. 3) There was no histochemical or ultrastructural evidence of cholesterol in BrM. 4) EC was histochemically detectable in the optic nerve and nerve fiber layer of 2 Diet1 animals, and the optic nerves contained 4-5X more EC than control animals. Conclusion: Diet-induced hypercholesterolemia is associated with prominent changes in the posterior rabbit eye in addition to well-described changes in cornea, sclera, and iris. The lack of UC and EC in BrM argues against the hypothesis that cholesterol accumulation in human BrM is attributable to direct deposition of plasma lipoproteins. Preliminary evidence of increased EC in optic nerve may indicate demyelination.
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