Do circulating microvesicles from patients with multiple sclerosis (MS) disrupt the blood-brain barrier (BBB)? (For Honors and MBiomedSc)
Breakdown of the blood brain barrier (BBB) precedes clinical symptoms of new lesions of MS and it is possible that high numbers of microvesicles in multiple sclerosis (MS) plasma are related to episodes of disruption of the BBB.
The integrity of BBB will be studied using an in vitro model examining lymphocyte transmigration across confluent monolayers of cultured endothelial cells. Human umbilical vein endothelial cells (HUVECs) are grown to confluent monolayers in tissue culture plates and peripheral blood lymphocytes added to each well and incubated for 2-4 h. The HUVEC layer is washed 5 times with saline media, then fixed and examined by phase-contrast microscopy. Cells beneath the monolayer appear phase dark while adherent cells above appear phase light. The number of adherent and migrated cells are counted to give an index of efficiency of migration. To assess if microvesicles impair the integrity of the endothelial monolayer, the migration assay will be performed both in the absence and presence of plasma containing known concentrations of platelet derived microvesicles. Meanwhile, the lysosomal β-hesosaminidase activity will be measured in platelet poor plasma from 20 MS patients and 20 controls using a standard colourimetric assay. The microvesicle counts, β-hesosaminidase activity and the impact on lymphocytes transendothelial migration will be analysed in correlation. Results could provide evidence for a mechanism by which peripheral blood leukocytes infiltrate to brain in MS.
Techniques involved include cell culture, ultra-centrifugation, flow cytometry, fluorescent microscopy and biochemistry.
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