2.1. Experimental design
New Zealand white male rabbits (1.5–2 years old and 3–5 kg) were assigned randomly to group 1 (n=6) fed with the normal chow and group 2 (n=6) fed with 2% (w:w) cholesterol (Harlan Teklad Global Diets, Madison, WI ) in chow for 12 weeks and then euthanized. At necropsy, animals were perfused with Dulbecco’s phosphate-buffered saline at 37 °C and brains were promptly removed and cut to yield two symmetrical hemispheres; one hemisphere for immunohistochemistry and one for Western blot and ELISA analyses.
2.2. Quantification of Aβ levels by ELISA
Aβ levels were quantified in the cortex and hippocampus of control and cholesterol-fed rabbits by ELISA using the Biosource kit as per the manufacturer’s protocol. Briefly, to measure the amount of Aβ 1–40 and Aβ 1–42, the wet mass of the brain tissue (100 mg) was homogenized thoroughly with 8X mass of cold 5 M guanidine HCl/50mM Tris HCl. The homogenates were mixed for 3–4 hours at room temperature. The samples were diluted with cold reaction buffer (Dulbecco’s phosphate buffered saline with 5% BSA and 0.03% Tween-20 supplemented with 1x protease inhibitor cocktail) and centrifuged at 16,000 × g for 20 minutes at 4°C. The supernatant was decanted, stored in ice until use, diluted at least 1:2 with standard diluent buffer, and quantified by calorimetric sandwich ELISA kits. For aggregated Aβ, the wet mass of the brain tissue (100 mg) was homogenized thoroughly with 10X volume of tissue extraction buffer (25 mM Tris-HCl and 150 mM NaCl, supplemented with protease inhibitors; pH 7.4). The samples were sonicated and centrifuged at 100,000 x g for 1 hour at 4°C. The supernatant was collected and diluted at least 1:2 in standard diluent buffer prior to analysis using the aggregated Aβ ELISA kit from Biosource. The quantity of Aβ in each sample was measured in duplicates. Protein concentrations of all samples were determined by standard BCA assay (Pierce). Aβ levels were normalized to total protein content in the samples.
2.3. Western blot analysis
Cortex and hippocampus from control and cholesterol-fed rabbit brains were homogenized in T-PER tissue protein extraction reagent (Thermo Scientific, Rockford, IL) supplemented with protease and phosphatase inhibitors. Protein concentrations were determined with BCA protein assay. Proteins (10μg) were separated in SDS-PAGE gels followed by transfer to a polyvinylidene difluoride membrane (Millipore, Bedford, MD) and incubation with antibodies to BACE1 (1:100, Chemicon International, Temecula, CA), RAGE (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), IDE (1:100, Chemicon International, Temecula, CA), and LRP-1(1:100, Biodesign International, Saco, Maine). β-actin was used as a gel loading control. The blots were developed with enhanced chemiluminescence (Immmun-star HRP chemiluminescent kit, Bio-Rad, Herculus, CA). Bands were visualized on a polyvinylidene difluoride membrane and analyzed by LabWorks 4.5 software on a UVP Bioimaging System (Upland). Quantification of results was performed by densitometry and the results analyzed as total integrated densitometric values (arbitrary units).
2.4. Confocal microscopy studies
Coronal frozen sections (14 μm) at the level of hippocampus and cortex were air-dried, fixed with cold acetone, blocked with 5% normal goat serum, and reacted overnight at 4°C with antibodies to BACE1 (1:250), RAGE (1:250), IDE (anti-mouse, 1:250, Signet laboratories), LRP-1 (1:500), or 6E10 (1:250, Signet laboratories Inc., Dedham, MA). According to the manufacturer, the region of the Aβ peptide where the 6E10 epitope resides is completely homologous between human and rabbit. Sections were then washed and incubated with secondary antibodies conjugated to Alexa fluor-488 (Molecular Probes, Inc., Eugene, OR) for one hour at room temperature in the dark and washed with PBS. The sections were then incubated in autofluorescence eliminator reagent (Chemicon International, Temecula, CA) for 5 minutes, washed with 70% ethanol, and mounted with vectasheild containing DAPI (Vector laboratories, Inc., Burlingame, CA). The sections were visualized with a Zeiss LSM 510 META confocal system coupled to a Zeiss Axiophot 200 inverted epifluorescence microscope. Imaging was performed with a 63X oil immersion objective. The dimensions of all images displayed on the monitor were 1024 × 1024 pixels. For single scanning detection of the fluorescence of Alexa-fluor(R) 488, a 488 nm Argon laser with wavelength filter, a 488/543 HFT and 545 NFT primary dichroic beam splitter, and a 505–530 nm band pass filter were used. The typical laser power at the sample was around 10% transmission. The DAPI cube was used for both viewing and taking images of the nucleus. The emission was a LP 420, which was combined with the mercury bulb to take the images.
2.5. Statistical Analysis
Quantitative data are presented as mean values ± SEM. The significance of differences between the control and cholesterol-fed group was assessed using the Student’s t test, with p < 0.05 considered statistically significant. Statistical analysis was performed with GraphPad Prism software 4.01.