Doctor Scientiae / The blood-brain barrier (BBB) is a restrictive interface located between the blood
circulation and the central nervous system (CNS), regulating the homeostatic
environment of the neuronal milieu, by controlling the permeability of the
cerebrovasculature. Currently, we cannot fully comprehend the regulatory features
and the complexity of BBB morphology to allow for intervention clinically. The
thesis consists of four publications. The methodology paper proposes a novel
experimental design to visualize the morphological architecture of immortalized
mouse brain endothelial cell lines (bEnd3/bEnd5). The brain endothelial cells
(BECs) were grown on cellulose matrices and fixed in 2.5 % glutaraldehyde in
preparation for visualization of the paracellular (PC) spaces between adjacent
BECs, employing high-resolution electron microscopy (HREM), with vested
interest in the morphological profile of the developing BEC. The second
publication addresses and reports on the nanosized detail of BEC monolayer
morphology utilizing high-resolution scanning electron microscopy (HR-SEM)
and published the first descriptions of the extrusion of a basement membrane from
developing in vitro BECs. Moreover, we categorized and discussed two types of
nanotubule (NT) development specific for the establishment of the BEC
monolayers. NTs can occur via nanovesicle extrusion onto the BEC membrane
surfaces, which fuse, forming tunneling NTs (TUNTs) between adjacent BECs.
Furthermore, cytoplasmic extensions of BEC membrane leading edges give rise to
tethering NT (TENTs), which result in overlapping regions across the PC spaces,
resulting in PC occlusion. BEC NT communication is illuminated in a third
publication utilizing immunofluorescence microscopy, which reports on the
molecular, cytoskeletal elements governing NT formation. This study shows, for
the first time, f-actin and α-tubulin cytoskeletal proteins extending between the
soma of the cells and NT cytoskeletal structures within an in vitro BBB model.
Thereafter, the effects depolymerizing agents, Cytochalasin D and Nocodazole,
were investigated on f-actin and α-tubulin cytoskeletal protein generation,functionality of NT morphology, cell division and permeability. For the first time,
we show that f-actin possesses an additional function, key to tight junction, plaque
protein organization. Moreover, it facilitates TENT formation, essential for
cytoplasmic projection across PC spaces. Conversely, α-tubulin facilitates known
functions: (i) transportation, (ii) cytokinesis, (iii) cellular division, and (iv)
possesses a novel function as the molecular cytoskeletal backbone of TENTs,
which facilitates BBB impermeability. A critical review evaluates past literature,
in light of the current findings emanating from this study. The review critiques the
concept of BEC cilia, which have been reported in the literature, comprised of
tubulin and actin, but at low-resolution. In the light of our novel observations,
nowhere in transmission electron microscopy do we observe cilia on the BECs,
we postulate that NTs have been misnamed and mischaracterized as cilia. The
thesis endeavors to elucidate the complexity of BEC nanostructures by examining
the emerging role of the nanoscopic landscape of BBB development and the
changing nature of BEC morphology, NT formation and associated
cytoarchitectural underpinnings governing NT morphology. The research study
attempts to, with a view to create new avenues for treating brain pathology,
revolutionize our interpretation of barrier-genesis on a nanoscale.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uwc/oai:etd.uwc.ac.za:11394/9085 |
| Date | January 2022 |
| Creators | Mentor, Shireen |
| Contributors | Fisher, David |
| Publisher | University of the Western Cape |
| Source Sets | South African National ETD Portal |
| Detected Language | English |
| Format | application/pdf |
| Rights | University of the Western Cape |
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