The blood-brain barrier (BBB) is a physical and biochemical barrier that precisely controls cerebral homeostasis. It also plays a central role in the regulation of blood-to-brain flux of endogenous and exogenous xenobiotics and associated metabolites. This is accomplished by molecular characteristics of brain microvessel endothelial cells such as tight junction protein complexes and functional expression of influx and efflux transporters. One of the pathophysiological features of ischemic stroke is disruption of the BBB, which significantly contributes to development of brain injury and subsequent neurological impairment. Biochemical characteristics of BBB damage include decreased expression and altered organization of tight junction constituent proteins as well as modulation of functional expression of endogenous BBB transporters. Therefore, there is a critical need for development of novel therapeutic strategies that can protect against BBB dysfunction (i.e., vascular protection) in the setting of ischemic stroke. Such strategies include targeting tight junctions to ensure that they maintain their correct structure or targeting transporters to control flux of physiological substrates for protection of endothelial homeostasis. In this review, we will describe the pathophysiological mechanisms in cerebral microvascular endothelial cells that lead to BBB dysfunction following onset of stroke. Additionally, we will utilize this state-of-the-art knowledge to provide insights on novel pharmacological strategies that can be developed to confer BBB protection in the setting of ischemic stroke.

The blood–brain barrier (BBB) is a critical regulator of CNS homeostasis. It possesses physical and biochemical characteristics (i.e. tight junction protein complexes, transporters) that are necessary for the BBB to perform this physiological role. Microvascular endothelial cells require support from astrocytes, pericytes, microglia, neurons, and constituents of the extracellular matrix. This intricate relationship implies the existence of a neurovascular unit (NVU). NVU cellular components can be activated in disease and contribute to dynamic remodeling of the BBB. This is especially true of microglia, the resident immune cells of the brain, which polarize into distinct proinflammatory (M1) or anti-inflammatory (M2) phenotypes. Current data indicate that M1 pro-inflammatory microglia contribute to BBB dysfunction and vascular “leak”, while M2 anti-inflammatory microglia play a protective role at the BBB. Understanding biological mechanisms involved in microglia activation provides a unique opportunity to develop novel treatment approaches for neurological diseases. In this review, we highlight characteristics of M1 proinflammatory and M2 anti-inflammatory microglia and describe how these distinct phenotypes modulate BBB physiology. Additionally, we outline the role of other NVU cell types in regulating microglial activation and highlight how microglia can be targeted for treatment of disease with a focus on ischemic stroke and Alzheimer’s disease.

The blood-brain barrier (BBB) allows the brain to selectively import nutrients and energy critical to neuronal function while simultaneously excluding neurotoxic substances from the peripheral circulation. In contrast to the highly permeable vasculature present in most organs that reside outside of the central nervous system (CNS), the BBB exhibits a high transendothelial electrical resistance (TEER) along with a low rate of transcytosis and greatly restricted paracellular permeability. The property of low paracellular permeability is controlled by tight junction (TJ) protein complexes that seal the paracellular route between apposing brain microvascular endothelial cells. Although tight junction protein complexes are principal contributors to physical barrier properties, they are not static in nature. Rather, tight junction protein complexes are highly dynamic structures, where expression and/or localization of individual constituent proteins can be modified in response to pathophysiological stressors. These stressors induce modifications to tight junction protein complexes that involve de novo synthesis of new protein or discrete trafficking mechanisms. Such responsiveness of BBB tight junctions to diseases indicates that these protein complexes are critical for maintenance of CNS homeostasis. In fulfillment of this vital role, BBB tight junctions are also a major obstacle to therapeutic drug delivery to the brain. There is an opportunity to overcome this substantial obstacle and optimize neuropharmacology via acquisition of a detailed understanding of BBB tight junction structure, function, and regulation. In this review, we discuss physiological characteristics of tight junction protein complexes and how these properties regulate delivery of therapeutics to the CNS for treatment of neurological diseases. Specifically, we will discuss modulation of tight junction structure, function, and regulation both in the context of disease states and in the setting of pharmacotherapy. In particular, we will highlight how these properties can be potentially manipulated at the molecular level to increase CNS drug levels via paracellular transport to the brain.

In this work, we examined the ability of gp120, a human immunodeficiency virus-1 (HIV-1) viral envelope glycoprotein, to trigger the innate immune response in astrocytes, an HIV-1 brain cellular target, and we investigated the functional expression of the ATP-binding cassette membrane transporter P-glycoprotein (P-gp) in primary cultures of rat astrocytes treated with gp120 or cytokines [tumor necrosis factor-␣ (TNF-␣), interleukin-1␤ (IL-1␤), and IL-6]. Standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium and D-mannitol uptake assays confirmed that HIV-1 96ZM651 gp120 treatment did not alter cell viability or membrane permeability. Semiquantitative reversetranscriptase polymerase chain reaction analysis and enzymelinked immunosorbent assay demonstrated increased TNF-␣, IL-1␤, and IL-6 mRNA and protein expression in cultures treated with HIV-1 96ZM651 gp120, suggesting in vitro activation of immune responses. Cytokine secretion was detected when CXCR4 but not CCR5 was inhibited with a specific antibody, implying that cytokine secretion is primarily mediated via CCR5 in astrocytes triggered with HIV-1 96ZM651 gp120. P-gp protein expression was increased in astrocyte cultures exposed to TNF-␣ (2.9-fold) or IL-1␤ (1.6-fold) but was decreased profoundly in the presence of IL-6 (8.9-fold), suggesting that IL-6 is primarily involved in modulating P-gp expression. In parallel, after HIV-1 96ZM651 gp120 treatment, immunoblotting analysis showed a significant decrease in P-gp expression (4.7-fold). Furthermore, the accumulation of two P-gp substrates, digoxin and saquinavir (an HIV-1 protease inhibitor), was enhanced (1.5-to 1.8-fold) in HIV-1 96ZM651 gp120-treated astrocyte monolayers but was not altered by P-gp inhibitors [e.g., valspodar (PSC833) and elacridar (GF120918)], suggesting a loss of transport activity. Taken together, these data imply that HIV-1 96ZM651 gp120 or cytokine treatment modulate P-gp functional expression in astrocytes, which may lead to complex drug-transporter interactions during HIV-1 encephalitis-associated immune responses.