Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

The overall goal of this procedure is to demonstrate the role of oxygen glucose deprivation as an in vitro ischemia reperfusion injury model for studying the blood brainin barrier dysfunction. This is accomplished by exposing brain endothelial cells grown in chamber slides to oxygen glucose deprivation by replacing the media with deoxygenated no glucose media and exposing them to a hypoxia chamber environment for two hours, the cells are then returned to normal conditions for an hour to achieve reoxygenation. After processing the cells immunofluorescent staining of Zola Occludin one and Rod Domine Phin labeling of filamentous actin is performed.

Cells are then imaged using a 60 x water immersion lens on a confocal microscope to obtain single optical slices for analysis, Demonstrating the procedure will be hem nega, a graduate student in my laboratory. To begin the procedure, have primary culture RBM eecs made from adult spra dolly rats on hand. Commercially available lines are also acceptable.

Cultivate the R BM EECS in 100 centimeter dishes coated with fibronectin culture them in rat brain endothelial cell growth medium, and change the medium every other day. Grow the cells until they are 80 to 90%confluent. Then gently wash the cells with five milliliters of PBS.

Next, detach the cells from the plate by first adding one milliliter of warm 0.25%tripsin EDTA solution and swirling the plate. Then incubate the cells for two to five minutes. Tap the side of the dish to increase the cell's, detachment and view the cells under a microscope to confirm their detachment.

To end the trypsin ization, add five milliliters of complete medium, then pipette out the medium with the detached cells and collect it into a 15 milliliter centrifuge tube. Centrifuge the media with the cells at 220 G for five minutes, aspirate and discard the supernatant. Then resuspend the cell pellet in three to five milliliters of fresh medium by gently flowing the cells through a five milliliter pipette.

Now count the cells and transfer 20 to 30, 000 to each well of a fibrin, actin coated eight well chamber slide. Of course, they could also be grown differently for other applications. Then incubate the chamber slide until the cells are confluent in preparation.

Set up and calibrate the hypoxia cell culture system. According to the manufacturer's instructions, the chamber environment should be 95%nitrogen, 5%carbon dioxide, and at 37 degrees Celsius. Now deoxy oxygenate glucose free DMEM by placing it in the hypoxia chamber for four to six hours later.

Begin stressing the cells by replacing their medium with the deoxygenated glucose free DMEM, and then placing the cells into the hypoxia chamber for two hours. After two hours, put the cells back onto rat brain endothelial cell complete medium. Then re oxygenate the cells by returning them to normal conditions for an hour.

Begin with exposing each well of R-B-M-E-C monolayers to 100 to 200 microliters of optimum reduced serum media and continue their incubation for an hour. Then wash the chamber slides three times using 100 to 200 microliters of PBS per well per wash. Fix the cells with 4%paraldehyde in PBS and incubate the slides for 15 minutes at room temperature and wash the cells off three times with PBS as before.

Now perme the cells by applying 100 to 200 microliters of 0.5%Triton X 100 in PBS to each well and let the plate incubate for another 15 minutes at room temperature. After the cells have been perme, apply the blocking solution and let this act on the cells for an hour at room temperature. Next, either perform immunofluorescence for ZO one protein or label F actin.

To conduct immunofluorescence of the cells for ZO one, incubate them with the primary antibody overnight at four degrees Celsius. The next day, wash the cells three times in PBS, such as when removing the fixative. Then incubate the cells in 100 to 200 microliters of fitzy tagged anti rabbit secondary antibody for an hour at room temperature to label the cells with r Domine Phin.

After applying the blocking solution for an hour, expose the cells to 100 to 200 microliters of R domine Phin in a one to 50 dilution with 2%B-S-A-P-B-S for 20 minutes. After the secondary antibody or R domine Phin incubation, wash the cells with PB S3 times again as before. Then remove the chambers by peeling off the elastic.

Proceed with mounting the slides using medium containing anti fade and DPI image. The cells with confocal microscopy in a single plane with a 60 x objective. Cells cultured on fibronectin coated chamber.

Slides were subjected to OGDR by placing them in a hypoxia chamber. As described. The cells were then processed for ZO one junctional.

Staining control cells showed continual junctional integrity while oxygen and glucose deprived cells had discontinuous junctions. Additionally, cytoskeletal changes were observed using aero domine foid in label to show F actin.Stress. Fiber formation control cells showed no or minimal F ACT in stress fiber formation while cells subjected to OGDR had increased stress fiber formation.

After watching this video, you should have a good understanding of how to perform oxygen, glucose deprivation and reoxygenation. This can be used as a ischemia reperfusion injury model to study blood-brain barrier, tight junction integrity and stress. Fiber formation.