Albert Sun⁽¹⁾, Bozena Draczynska-Lusiak⁽²⁾, Grace Sun^{(3)
(1)(2)(3)}Department of Pharmacology. University of Missouri - Columbia. United States

MATERIALS AND METHODS

Primary cortical neuronal culture

Mixed neocortical neuronal cell cultures, containing mainly neurons and a small amount of glial cells, were prepared from fetal rats at 18-19 days of gestation using protocols as described by Cheng and Sun (1994). Dissociated cortical cells were plated in Eagle’s minimal essential medium (MEM) supplemented with 20 mM glucose, 2 mM glutamine and 10% fetal bovine serum. When the culture cells reached confluency, non-neuronal cell division was arrested by exposing the culture to 10 mM cytosine arabinofuranoside for 1-3 days. Cultures were maintained at 37^oC in a humidified incubator containing 5% CO₂ and atmospheric oxygen. In most instances, cells were used for experiments after maintaining in culture for 10-14 days.

Preparation of aggregated beta amyloid peptide (Ab)

Preparation and aggregation of Ab was similar to that descrebed by Hu et al. (1998). Briefly, 1 mg of Ab ( 1-42 ) (BACHEM, King of Prussia, PA) was first dissolved in 100 ml dimethysulfoxide (DMSO) and then diluted 10 fold with sterile distilled water. Ab suspension was then aged at room temperature for 4 days. To verify aggregation, an aliquot of the aged Abwas examined under an inverted microscope (Nikon Diaphot 300, Nikon Corp. Melville, NY). Aggregated Abs formed a turbid solution and when examined under the microscope, aggregates of beta sheet conformation could be observed.

Preparation of lipoproteins with brain lipids (BLPs).

For extraction of lipids, rat brain was homogenized with phosphate-buffered saline (PBS) and partitioned with 4 vol of chloroform-methanol (2:1, v/v). After evaporating the solvents under nitrogen, the lipids were hydrated in a medium composed of 0.1 M KCl, 0.01 M Tris-HCl (pH 8.0) and bovine serum albumin (BSA, 1mg/ml). This mixture was sonicated and purified as described by Rensen et al. (1997). Briefly, BLPs were concentrated by density gradient ultracentrifugation using NaBr/EDTA density solution at 43,500 rpm for 18-22 hr at 4^oC. The lipoprotein band was isolated by aspiration. BLPs were then divided into two fractions, one was subjected to overnight oxidation with 0.1 M FeCl₃ (Evans et al. 1997). Both native and oxidized BLP were subjected to dialysis overnight against a phosphate buffered saline-EDTA solution. ApoE e3 and apoE e4 were initially gifts from Dr. Holtzman (Washington University, St. Louis, MO) and later purchased from PanVern Corp, Madison, WI). Native and oxidized BLPs (0.5 mg/ml) were incubated with apoE (10 mg/ml) at 37^o for 30 min to obtain the apoE3- or apoE4-enriched BLPs.

Methods to assess uptake of BLPs by primary cortical neurons

1. Confocal microscopy using the fluorescent probe 3,3-dioctadecylindocarbocyanine (Di-I): Native and oxidized BLPs were labeled with 50 ml of Di-I (3mg/ml) at 37^oC for 15 hr and dialyzed with saline solution containing 0.01% EDTA according to procedures described by Pitas et al. (1981). Neuronal cells were plated on the coverslips and treated with the Di-I labeled native or oxidized BLPs for 3-4 hr. After incubation, the cells were washed five times with PBS containing 2 mg/ml BSA and once with PBS, and then fixed for 30 minutes with 3% paraformaldehyde in PBS (pH 7.4). Confocal and fluorescence microscopy was performed with epifluorescent illumination using a rhodamine filter package.
2. Labeling with [¹⁴C]cholesterol: In this preparation, a known amount of [¹⁴C] cholesterol was added to the lipid extract and the preparation of BLP was performed as described above. Both native and oxidized BLPs were enriched with apoE e3 or apoE e4, and were incubated with primary neurons for various time periods. After incubation, neuronal cells were washed two times with PBS, suspended in 0.5 ml of water and the amount of radioactivity incorporated into the cells was counted using a Beckman’s 5800 liquid scintillation counter (Beckman, Sunnyvale, CA).

Lactate dehydrogenase (LDH) release assay.

Neuronal cell death was determined by assessing the release of lactate dehydrogenase (LDH) into the medium using the spectrophotometric method described by (Cheng et al, 1994). Briefly, 0.2 ml of the culture medium was added to a phosphate-buffered pyruvate (0.6 mM, pH 7.5) (Sigma Chem Corp., St. Louis, MO) and followed by addition of NADH to initiate the reaction. The assays were carried out at 25^oC and changes of absorbency at 340 nm were measured using a Beckman spectrophotometer equipped with a kinetic program.

Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Neuronal cell survival was also assessed by the MTT reduction assay protocol. This method is based on the reduction of the yellow MTT (Sigma Chem Corp., St. Louis, MO) to its blue formazan product by dehydrogenases in the mitochondria. The amount of formazan produced is proportional to the number of living cells (Li and Sun, 1999). Cells were treated with native and oxidized BLPs and after an appropriate time (24-48 hr), MTT was added (0.5mg/ml) and incubated for 3 hr at 37oC. Upon removal of the media, blue formazan produced was solubilized in DMSO and measured with a spectrophotometer at 540 nm. Results were expressed as percent of control.

Discussion Board

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[ABSTRACT] [INTRODUCTION] [MATERIALS AND METHODS] [RESULTS] [IMAGES] [DISCUSSION] [ACKNOWLEDGEMENT] [REFERENCES] [Discussion Board]

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Albert Sun, Bozena Draczynska-Lusiak, Grace Sun
Copyright © 1999-2000. All rights reserved.

Last update: 31/1/2000