Vesicular transport across vascular blood-neural barriers

Abstract

In order to establish an isolated environment for the nervous system, the blood-brain barrier (BBB) has to separate the neuronal parenchyma from the cardiovascular circulatory system. It predominantly acts on the endothelial cells; however, the neurovascular unit that includes the neuronal cell, astrocytes and pericytes with the interaction of functional and apposition, is vitally essential for the regulation process. In the BBB, the presence of TJs and low level of transcytosis make the barrier strictly impermeable. In pathological conditions, leakage can occurs in both pathways, via pracellular (TJs) or/and transcellular (transcytosis). In transcellular leakage, caveolae is likely to be involved as evidence from previous literature. However, caveolar role in leakage is still disputed. The psychoactive drug methamphetamine (METH), at low concertation, induces leakage in the BBB exclusively via caveolae. This has been correctly investigated in the host laboratory by a method involving in situ carotid dual perfusion and delivery of compounds to Wistar rat brains post-mortem and followed by Miles leakage assays. Previous work in the lab, electron microscope (EM) has been used to investigate caveolar transport induced by METH. To this point, this has permit to visualise tracer filled caveolae. However, mechanistic study including antibodies would necessitate more complex protocols (e.g. cryo-immuno-EM), which is limited to very few antibodies. A relatively new technique called expansion microscopy (ExM) physically expand the sample to achieve a nanoscale imaging. ExM would form an alternative carrying a number of advantages over EM, for instance workability, in theory, any antibodies that compatible in 3

immunohistochemistry, and an easier and less time consuming preparation. ExM can also achieve a resolution of ~65 nm, hence possibly allowing the necessary resolution to study tracer filled caveolae during BBB leakage. However, ExM never been established in the department, thus adopting ExM protocol might carry limitations to achieve a suitable expansion with preserved fluorescent. After numerous optimization of the technique, including immunostaining, anchoring, and digestion and so on, we managed to achieve the targeted expansion factor (x4.5) as well as preserved vascular integrity. However, further optimizations are needed to preserve a strong signal retention of cavolin-1 staining after expansion. Moreover, the implementation of higher magnifications images using a water immersion lenses. Future studies, will also focus on the mechanism of caveolar role in transportation as well as signalling at the BBB.