Our research is focused on the placenta brain communication. We are exploring whether a small extracellular vesicles can disrupt the blood brain barrier, and if that is the potential underlying mechanism associated with cellular vascular complication observed in women with preeclampsia. We have been previously shown that plasma of women with preeclampsia can impair the blood brain barrier.
However, the nature of this potential harmful factor present in the plasma is unknown. Research from our laboratory showed that extracellular vesicles derived from the plasma of women with preeclampsia or placentas cultured in hypoxia impairs the blood brain barrier. The underlying mechanism of how placental extracellular vesicles impairs the blood brain barrier need further investigation that include analysis of the container of extracellular vesicles, such as a specific microRNAs that control the expression of digestion proteins in brain endothelial cells.
To begin extracting explants of the human placenta sample using sterilized forceps, carefully remove the clots from the basal plate of the placenta. Extract small explants until a final tissue extraction of about 10 grams is obtained. After washing and resuspending the explants in culture media, split the resuspended explants into two 100 millimeter culture dishes.
Place one of the dishes in standard normoxic conditions at 37 degrees Celsius with 8%oxygen and 5%carbon dioxide. Place the other dish in a hypoxic chamber with 1%oxygen. After culturing the explants under two different conditions for 18 hours, proceed to harvest the conditioned media of the explants in a 15 milliliter tube.
Collect the culture media from the normoxic and hypoxic explant cultures in separate tubes. Perform sequential centrifugation of the harvested conditioned medium at room temperature. After each centrifugation using a 20 to 100 microliter pipette, carefully collect the supernatant while discarding the pellet.
Perform one additional centrifugation on the last collected supernatant. Resuspend the obtained pellet containing placental small extracellular vesicles or SEVs in 500 microliters of PBS and pass the suspension through a 0.22 micron filter. The placenta derived SEVs are now ready to be injected into the mouse.
To perform small extracellular vesicles or SEVs injections into the mouse, use the SEVs isolated from human placental explants, cultured under hypoxic and normoxic conditions. Dilute 200 micrograms of placental SEVs in phosphate buffer at pH 7.4 up to a final volume of 70 microliters. Before the injection, evaluate the animal's wellbeing using the RMCBS scale.
Then using an insulin syringe with a 30 gauge needle inject the solution into the external jugular vein of the properly anesthetized mouse. After injection apply light pressure to the injected area for 15 seconds with a dry cotton swab. Evaluate mouse wellbeing using the RMCBS scale at three, six, 12, and 24 hours after injection.
For analysis of Evans blue extravasation, prepare 2%Evans blue dye solution in PBS. 24 hours after injecting SEVs using retroorbital access, inject Evans blue solution into the properly anesthetized mouse. Once Evans blue is circulated in the animal body, perform intracardiac perfusion on the anesthetized mouse with about three milliliters of saline solution to remove the dye from the circulation.
After fixing the mouse with an intracardiac infusion of 4%paraformaldehyde in PBS and carefully extracting the brain, weigh and photograph it. Following the demonstrated procedure, extract the brains from the animals in each group using a stereo zoom microscope, visualize the captured images of the dissected brain slices. Use the captured images to quantify Evans blue extravasation using Image J software.
Contrary to mice injected with SEVs from normoxic placentas, mice injected with SEVs from hypoxic placentas showed a progressive decline in the neurological score until 24 hours post-injection. Brains from mice injected with SEVs from hypoxic placentas had higher Evans blue extravasation than brains from animals injected with SEVs from normoxic placentas. Brains of mice injected with SEVs from hypoxic placentas showed reduced amounts of clot in five protein, which is critical for tightness of the blood-brain barrier.
All these results indicated disruption of the blood-brain barrier introduced by SEVs from hypoxic placentas.
