LE3 .A278 2018

2018

Wilson, Brian

Acadia University

Bachelor of Science

Honours

Biology

Biology

Endothelial cells and paracellular tight junctions of the blood-brain barrier (BBB) restrict the passage of most compounds between the vascular system and brain parenchyma. Tight junctions are modulated by cadherin interactions joining the vascular endothelial cells of capillaries via adherens junctions. Under neuropathological conditions, i.e., cancer, these tight junctions become compromised, leading to alterations of BBB permeability. Moreover, the restrictive permeability of the BBB is an obstacle for the delivery of potential treatments. Primary cultures of endothelial cells have been used to develop a better understanding of the molecular mechanisms regulating permeability of the BBB, however evidence suggests a lack of growth factors in monoculture models creates uncharacteristic leakiness of in-vitro tight junctions. As the secretion of these factors is thought to be of astrocytic or microglial origin, this study attempted to quantify the expression of sonic hedgehog (SHH), wingless (WNT) and norrin (NDP) proteins in the sub-ventricular zone (SVZ), hippocampus and cerebellum respectively. Organotypic brain slice cultures were prepared and cultured for two weeks, then fixed in paraformaldehyde for immunofluorescence detection, or flash-frozen with liquid nitrogen for molecular studies using RT-qPCR. Western blotting and immuno-labelling of the SVZ showed high levels of expression of SHH protein. Hippocampal and cerebellar slices showed high levels of expression of WNT-7a and NDP immunofluorescence, respectively. Furthermore, we are optimizing primers for shh, wnt-7a, and ndp gene sequences for use in RT-qPCR studies of gene expression.

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https://scholar.acadiau.ca/islandora/object/theses:2621