Search

The blood-brain barrier (BBB) is increasingly being recognized as a site of special scientific importance. Numerous models of the BBB have been constructed over the past years with increasingly mechanistic studies of fundamental questions of cell biology and neuroimmunology. However, there has been a limiting factor of not being able to perform real-time studies of BBB function utilizing 3D models. Equally, real-time models have been limited mainly to 2D models comprised solely of endothelial cells (ECs). To measure changes in the electrical resistance across a BBB model, when adding inflammatory or stem cells which will interact with co-cultured glial cells has, to date, been beyond the capabilities of models. We have cultured an inverted BBB model with ECs on electrodes which are on the lower surface of xCELLigence Cell Invasion Migration plates. Glial cells were cultured in the basal well with foot processes extending through the filters to make contact with the ECs. SIV-infected macrophages decreased electrical resistance of the EC monolayer when added to the "parenchymal" face of the model. We present a novel inverted blood-brain barrier model that allow real time analyses of endothelial cell adhesion during modeled neuroinflammation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Expression of tight junction proteins between brain microvascular endothelial cells (BMECs) of the blood-brain barrier (BBB) is lost during development of human immunodeficiency virus (HIV) encephalitis (HIVE). Although many studies have focused on the strains of virus that induce neurological sequelae or on the macrophages/microglia that are associated with development of encephalitis, the molecular signaling pathways within the BMECs involved have yet to be resolved. We have previously shown that there is activation and disruption of an in vitro BBB model using lentivirus-infected CEMx174 cells. We and others have shown similar disruption in vivo. Therefore, it was of interest to determine if the presence of infected cells could disrupt intact cerebral microvessels immediately ex vivo, and if so, which signaling pathways were involved. The present data demonstrate that disruption of tight junctions between BMECs is mediated through activation of focal adhesion kinase (FAK) by phosphorylation at TYR-397. Inhibition of FAK activation is sufficient to prevent tight junction disruption. Thus, it may be possible to inhibit the development of HIVE by using inhibitors of FAK.

The blood-brain barrier (BBB) plays a critical role in normal physiology of the central nervous system by regulating what reaches the brain from the periphery. The BBB also plays a major role in neurologic disease including neuropathologic sequelae associated with infection by human immunodeficiency virus (HIV) in humans and the closely related simian immunodeficiency virus (SIV) in macaques. In this review, we provide an overview of the function, structure, and components of the BBB, followed by a more detailed discussion of the subcellular structures and regulation of the tight junction. We then discuss the ways in which HIV/SIV affects the BBB, largely through infection of monocytes/macrophages, and how infected macrophages crossing the BBB ultimately results in breakdown of the barrier.

It is known that there is disruption of the blood-brain barrier during terminal AIDS encephalitis in both human immunodeficiency virus (HIV)-infected humans and simian immunodeficiency virus (SIV)-infected rhesus macaques. Much, although by no means all, of the neuropathological findings of HIV and SIV infection involves accumulation of monocytes/macrophages that have likely crossed the blood-brain barrier (BBB). There is no convincing, rigorous, demonstration of HIV (or SIV) infecting endothelial cells in vivo. However, this is not to say that HIV infection would not have any effects on the physiology of microvascular brain endothelial cells. Because of the elaborate nature of cerebral microvessels, previous studies of cerebral endothelial cells have been constrained by sectioning artifacts. Examination of freshly isolated cerebral microvessels allows investigation of extended lengths of vessels (>150 mum) without sectioning artifacts. These studies determine the changes in the expression of the tight junction protein zo-1 protein on the endothelial cells of cerebral capillaries at terminal acquired immune deficiency syndrome, demonstrating that there is a decreased expression of zo-1 protein over extended lengths of microvessels.

The primary cell infected during acute HIV neuropathogenesis is the monocyte-derived macrophage. We have demonstrated that there is activation of the BBB (blood-brain barrier) during acute viral infection and at terminal AIDS. However, it has never been determined if there is a requirement for the virus to be carried through the BBB or how these Trojan horses would be induced to cross the BBB. We added SIVmac251-infected (SIV is simian immunodeficiency virus) mononuclear cells (and their supernatants) to the luminal or abluminal compartment of our BBB model. There was activation of both sides of the BBB model, only if viral-infected cells were added to the luminal compartment, as opposed to the addition of cell-free supernatants. This suggests that cell-associated virus is essential for the activation of the BBB. This, in turn, would be expected to lead to further infiltration of cells capable of viral infection. VCAM-1 (vascular cell adhesion molecule 1) staining revealed, for the first time, that there is an absolute requirement for virally infected cells, and not just the presence of virus for the activation of the BBB.

It has never been determined if activation of the blood-brain barrier (BBB) during simian immunodeficiency virus/human immunodeficiency virus (SIV/HIV) infection is a function of high levels of circulating virus or if the virus has to be within a cell capable of crossing the BBB to activate it. In vitro models of the BBB are becoming recognized as an acceptable method for determining the cellular events associated with HIV neuroinvasion. Cell free virus (when added in the physiologically relevant lumen) although capable of activating the endothelial cells of our in vitro BBB did not activate astrocytes beneath. SIVmac251-infected CEMx174 cells, however, were capable of activating both components of the BBB model. Here we demonstrate that an in vitro model of the BBB can be activated in a physiologically relevant manner, that SIV requires to be cell-associated and that endothelial cells of the BBB are not the only components that are activated during SIV neuroinvasion.

The blood-brain barrier (BBB) has been modeled in vitro in a number of species, including rat, cow and human. Coculture of multiple cell types is required for the correct expression of tight junction proteins by microvascular brain endothelial cells (MBEC). Markers of inflammation, especially MHC-II, and cell adhesion molecules, such as VCAM-1, are not expressed on the luminal surface of the barrier under resting conditions. The rhesus macaque model has been used to study early events of HIV-neuropathogenesis in vivo, but a suitable in vitro model has not been available for detailed mechanistic studies. Here we describe an in vitro rhesus macaque blood-brain barrier that utilizes autologous MBEC and astrocytes. We believe that this model is highly relevant for examining immunological events at the blood-brain barrier and demonstrate its potential usefulness for examining early events in AIDS neuropathogenesis.