After maturation, maintenance of the BBB and preservation of brain homeostasis is largely dependent on adequate perfusion to neuronal tissue and neuronal signaling to cerebral vessels, a process known as hyperaemia ( Attwell et al., 2010). A study carried out using rat microvascular endothelial cells and neuronal progenitor cells, showed that in the presence of neural progenitor cells, endothelial cells established regular TJ formation including: claudin 5, zonula occludens (ZO-1) and occludin ( Weidenfeller et al., 2007). Further to this, early neuronal signaling has been shown to be essential for the maturation of the BBB, specifically, tight junction (TJ) organization. Specifically, Wnt signaling in early CNS development is responsible for vascular stabilization and angiogenesis ( Liebner et al., 2008). Neural progenitor cells present in the ventricular neuroepithelium have been shown to aid endothelial cell recruitment during early BBB development, which is largely governed by the Wnt signaling pathway ( Risau et al., 1986). Several studies have also highlighted the roles of neurons and glia in BBB development and maintenance. Thus, pericytes play a large role in cerebral vascular function under normal physiological conditions as well as vascular dysfunction in hypoxia. These alterations in pericyte morphology, coupled with an upregulation in the expression of adhesion molecules and leukocyte integrin ligands, contribute to the extravasation of peripheral leukocytes into the brain following ischaemic insult ( Pieper et al., 2013). In pathologies such as ischaemic stroke, large gaps can develop between adjacent pericytes, increasing barrier permeability and vessel leakage. Pericytes have also been shown to secrete angiogenic factors such as vascular endothelial growth factor (VEGF), that support endothelial cell survival and proliferation ( Darland et al., 2003). As well as offering mechanical support, they also regulate vessel contractility, endothelial proliferation, blood flow and angiogenesis ( Bergers and Song, 2005 Dore-Duffy, 2008). In the CNS, pericytes are present at a higher ratio to HBMECs in the brain compared to the periphery and recent studies have shown the extensive role of pericytes in BBB development and maintenance ( Kacem et al., 1998 Armulik et al., 2011 Sweeney et al., 2016). Pericytes contribute 22–32% of the cerebral vasculature and together with vascular smooth muscle cells and endothelial cells they maintain vascular function ( Martini and Bartholomew, 2017). Neurons and microglia also contribute to the maintenance of the BBB and form what is known as the neurovascular unit (NVU) ( Abbott et al., 2006).
Structurally, the BBB is comprised of specialized brain microvascular endothelial cells (HBMECs), perivascular cells (pericytes) and astrocytes ( Abbott et al., 2006, 2010). The blood brain barrier (BBB) is a unique interface that separates the peripheral blood supply and neuronal tissue. We consider that this model is more representative of the interactions at the neurovascular unit than other transwell models and is a useful method to study BBB physiology. These data highlight the important role that neurons play in response to ischaemia, particularly how they contribute to BBB maintenance and breakdown. Our data also show that a four cell model responds faster to the barrier tightening effects of glucocorticoid dexamethasone, when compared to a two cell and three cell model. Compared to a two and three cell model, we show that when neurons are added to HBMECs, astrocytes and pericytes, BBB integrity was more sensitive to oxygen-glucose deprivation evidenced by increased permeability and markers of cell damage. We show that using a larger membrane pore size (3.0 μM) there is an improved connection between the endothelial cells, astrocytes and pericytes. Our novel model uses only primary human cells and includes four of the key cells of the BBB: astrocytes, pericytes, brain microvascular endothelial cells (HBMEC) and neurons.
It is critical that effective in vitro models are developed to model the in vivo environment to aid in clinically relevant research, especially regarding drug screening and permeability studies. Structural alterations and breakdown of the blood brain barrier (BBB) is often a primary or secondary consequence of disease, resulting in brain oedema and the transport of unwanted substances into the brain.
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