Fig. 2.

Summary of in vitro models of the brain and BBB. This diagram summarises the increasing complexity of the models and indicates the possible experimental approaches to interrogate the mechanisms via which P. falciparum-infected red blood cells disrupt the BBB and to identify and test potential adjuvant therapeutics. (A) Boyden chambers with mono-/co-culture systems allow for the measurement of barrier integrity in response to iRBC exposure. By activating the endothelial layer, transmigration of iRBCs/leukocytes is measurable. (B) The vasculature is under constant fluid flow and this can be mimicked using 2D or 3D microfluidic chips. Pumps generate shear stress recapitulating that found within the microvasculature. Adhesion under flow conditions can be quantified, as can the effect of antibodies against the parasite-derived PfEMP1. To assess barrier permeability, TEER and solute transport using fluorescent tracers can also be measured. Alterations of endothelial proteins such as actin (red) or the tight-junction protein ZO-1 (green) can be visualised via immunofluorescence. (C) The complexity of the BBB model can be increased by generating BBB organoids. These consist of astrocytes, pericytes and an outer layer of endothelial cells. This allows for the generation of a functional BBB and adhesion of iRBCs. Owing to the induction of swelling by specific iRBCs, which are capable of entering and migrating into the organoids, they also serve as a model for the investigation of neuroprotective agents. (D) The most complex model is the cerebral organoid. Although lacking endothelial cells/vascularisation, cerebral organoids offer a unique opportunity to investigate neurodevelopment in response to parasite exposure and the toxicity of parasite-derived products. BBB organoid and primary human brain endothelial monolayer images courtesy of Y.A., University of Copenhagen. Cerebral organoid image ©IMBA reused with permission (Lancaster et al., 2013). This image is not published under the terms of the CC-BY license of this article. For permission to reuse, please see Lancaster et al. (2013). BBB, blood–brain barrier; iRBC, infected red blood cell; TEER, transepithelial electrical resistance; ZO-1, zonula occludens protein 1.