This study investigates the effects of hypoxia on rat brain microvascular endothelial cells (RBMECs) and the subsequent disruption of tight junctions. The methodology involves manipulating the cellular environment to simulate conditions that affect the blood-brain barrier.
Take a chambered slide with a monolayer culture of rat brain microvascular endothelial cells, or RBMECs.
The cells are connected by tight junction proteins linked to the actin cytoskeleton, mimicking the endothelial layer of the blood-brain barrier.
Replace the medium with a deoxygenated glucose-free medium, then place the slide in a hypoxia chamber.
The unavailability of oxygen and glucose inhibits ATP synthesis. The remaining ATP is broken down into nucleotide derivatives.
ATP depletion hinders calcium pumps, elevating intracellular calcium that activates oxidoreductase enzymes.
Next, replenish with an oxygenated medium, then place the slide in an oxygenated incubator. The activated oxidoreductases utilize the reintroduced oxygen to degrade the nucleotide derivatives, generating reactive oxygen species or ROS.
ROS disrupts the interactions between tight junction proteins and induces signaling pathways that remodel the actin cytoskeleton into stress fibers, weakening cell-cell interactions.
The monolayer with disrupted intercellular interactions is ready for analysis.
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, deoxygenate glucose-free dmem by placing it in the hypoxia chamber for 4 to 6 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.
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