Elevated cholesterol is a major risk factor for cardiovascular and neurodegenerative disease. Our protocols provide valuable tools for studying both physiological and mechanistic consequences of hypercholesterolemia. These procedures can be performed using basic lab equipment, and are applicable to cells, tissues, and Xenopus oocytes.
Cholesterol is a major component of cellular membranes throughout the body. These techniques can be utilized to study the impact of elevated levels of cholesterol in any cell type. Dissecting the arteries is the most difficult step.
Carefully remove the artery from the brain, without stretching or damaging the vascular tissue. Lipids are very delicate, and working with them is more of an art than a science. Video demonstration provides a guide for learning how to handle lipids carefully.
To prepare the cholesterol saturated methyl-beta-cyclodextrin solution, first add 064 grams of methyl-beta-cyclodextrin to 10 milliliters of PBS, stirring the solution with a stir bar, to ensure that the methyl-beta-cyclodextrin fully dissolves. Next, add 0024 grams of cholesterol powder to the flask, and stir the solution vigorously, using a spatula to break up as many cholesterol chunks as possible. When nearly all the cholesterol has been dissolved, seal the flask with at least two layers of paraffin film.
And shake the flask at about 30 oscillations per minute in a 37 degree Celsius water bath overnight. After eight to 16 hours, cool the solution to room temperature, before filtering the mixture through a 22 micrometer polyethersulfone syringe filter into a class bottle. For mammalian cerebral artery enrichment, harvest the brain from a 250 to 300 gram Sprague Dawley rat into a beaker of PBS on ice, before transferring the brain into a waxed dissection bowl, under a dissecting microscope, containing just enough PBS to submerge the tissue.
Secure the brain with two to three pins, making sure they do not penetrate blood vessels. And use sharp forceps and small surgical scissors to gently dissect the cerebral arteries and their branches that form the Circle of Willis at the base of the brain. Next, briefly rinse up to one centimeter long artery segments in PBS, before incubating the rinsed segments in enough cholesterol enriching solution to cover the tissues.
To determine any alterations in cholesterol level, use a fresh bottle of filipin powder to prepare a 10 milligram per milliliter stock solution of the dye in dimethyl sulfoxide, and wash the cholesterol enriched tissues with three five minute washes in fresh PBS. After the last wash, fix the artery segments in 4%paraformaldehyde for 15 minutes on ice, protected from light, before permeabilizing the samples in 5%Triton in PBS for 10 minutes at room temperature. At the end of the incubation, wash the tissues with three five minute washes in PBS on a shaker, before adding the samples to a 25 microgram per milliliter final concentration of filipin dye solution, for one hour at room temperature, protected from light.
At the end of the incubation, wash the artery pieces three times as demonstrated, followed by a brief rinse with distilled water. After the last wash, use a lab tissue to absorb any excess liquid, and mount the arteries onto a slide using an appropriate mounting medium. Carefully cover the arteries with a cover slip, taking care to avoid rolling or twisting of the tissue, and let the slide dry for 24 hours at room temperature, protected from light.
When the mounting medium is dry, seal the cover slip edges with clear nail polish, and allow the polish to dry for 10 to 15 minutes. Then, image the tissue with an excitation of 340 to 380 nanometers, and an emission of 385 to 470 nanometers. To prepare the phospholipid based dispersion, combine 200 microliters of 10 milligram per milliliter chloroform dissolved lipid solution, L-alpha-phosphatidylethanolamine, 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and cholesterol in a 12 milliliter glass tube.
Evaporate the chloroform in the hood under a stream of nitrogen, before suspending the lipids in 800 microliters of buffered 150 millimolar potassium chloride and 10 millimolar Tris HEPES solution. Then seal the tube with paraffin film, and gently sonicate the tube contents at 80 kilohertz for 10 minutes, until a milky mixture is formed. For cholesterol enrichment of Xenopus oocytes, use sharp forceps to disrupt the ovarian sac from a female Xenopus laevis frog in multiple regions, and place the ovary chunks into a 60 millimeter plate with five milliliters of calcium free ND96 supplemented with 5 milligrams per milliliter of collagenase.
Shake the tissue on an orbital shaker at 60 oscillations per minute for 15 minutes at room temperature. At the end of the incubation, use a transfer pipette with a wide tip to vigorously pipette the oocyte containing solution five to 10 times to isolate the individual oocytes, and quickly rinse the dark oocyte solution with fresh calcium free ND96 until the solution becomes transparent. Next, transfer 90 microliters of the cholesterol enriched phospholipid based dispersion into one well of a 96 well plate, and add up to six oocytes to the well with as little medium as possible.
Place the 96 well plate onto a three dimensional platform rotator for five to 10 minutes. Then add a drop of ND96 to the well, and transfer the cholesterol enriched oocytes to a 35 millimeter plate containing ND96 for their immediate analysis. Here, an example of an imaged cerebral artery smooth muscle layer demonstrates the concentration dependent increase in filipin associated fluorescence obtained upon tissue enrichment with increasing concentrations of cholesterol.
Notably three hours subsequent to the treatment with the methyl-beta-cyclodextrin cholesterol complex the cholesterol levels decreased by approximately 50%compared to their level immediately after the enrichment. While a one hour incubation time is commonly used to enrich the tissues and cells with cholesterol during this approach, five minutes of incubation is usually sufficient to achieve a statistically significant increase in cerebral arterial cholesterol content as determined by cholesterol oxidase based biochemical assay. While no significant change is observed in cholesterol levels in Xenopus oocytes enriched with control phospholipid based dispersions lacking cholesterol, cholesterol levels increase significantly after only five minutes of treatment with the phospholipid based dispersions that include cholesterol, and remain at this level when the incubation time is increased to 60 minutes.
The effectiveness of the cyclodextrin based approach for enriching cells is also demonstrated in neurons freshly isolated from the CA1 region of the hippocampus. Indeed, incubation of the neurons in methyl-beta-cyclodextrin saturated with cholesterol for 60 minutes results in an over two times increase in cholesterol levels, as assessed by the filipin associated fluorescence. Overall, the two most important steps for these procedures are preparing the cholesterol enrichment mixture and ensuring the integrity of the arterial tissue.
Following cholesterol enrichment, one can study the function of the proteins that are affected by hypercholesterolemia. The ability to enrich cells with cholesterol facilitated the study of elevated cholesterol levels in cardiomyocytes and neurons. The use of oocytes allowed us to investigate how cholesterol binds to ion channels.
A lab coat and gloves she always be worn for this protocol. Chloroform and paraformaldehyde should be used under a fume hood and be discarded as hazardous waste.
