No abstract

Neuroinflammation is involved in the onset or progression of various neurodegenerative diseases. Initiation of neuroinflammation is triggered by endogenous substances (damage-associated molecular patterns) and/or exogenous pathogens. Activation of glial cells (microglia and astrocytes) is widely recognized as a hallmark of neuroinflammation and triggers the release of proinflammatory cytokines, leading to neurotoxicity and neuronal dysfunction. Another feature associated with neuroinflammatory diseases is impairment of the blood-brain barrier (BBB). The BBB, which is composed of brain endothelial cells connected by tight junctions, maintains brain homeostasis and protects neurons. Impairment of this barrier allows trafficking of immune cells or plasma proteins into the brain parenchyma and subsequent inflammatory processes in the brain. Besides neurons, activated glial cells also affect BBB integrity. Therefore, BBB dysfunction can amplify neuroinflammation and act as a key process in the development of neuroinflammation. BBB integrity is determined by the integration of multiple signaling pathways within brain endothelial cells through intercellular communication between brain endothelial cells and brain perivascular cells (pericytes, astrocytes, microglia, and oligodendrocytes). For prevention of BBB disruption, both cellular components, such as signaling molecules in brain endothelial cells, and non-cellular components, such as inflammatory mediators released by perivascular cells, should be considered. Thus, understanding of intracellular signaling pathways that disrupt the BBB can provide novel treatments for neurological diseases associated with neuroinflammation. In this review, we discuss current knowledge regarding the underlying mechanisms involved in BBB impairment by inflammatory mediators released by perivascular cells.

The blood-brain barrier (BBB) is highly restrictive of the transport of substances between blood and the central nervous system. Brain pericytes are one of the important cellular constituents of the BBB and are multifunctional, polymorphic cells that lie within the microvessel basal lamina. The present study aimed to evaluate the role of pericytes in the mediation of BBB disruption using a lipopolysaccharide (LPS)-induced model of septic encephalopathy in mice. ICR mice were injected intraperitoneally with LPS or saline and were sacrificed at 1, 3, 6, and 24 h after injection. Sodium fluorescein accumulated with time in the hippocampus after LPS injection; this hyperpermeability was supported by detecting the extravasation of fibrinogen. Microglia were activated and the number of microglia increased with time after LPS injection. LPS-treated mice exhibited a broken basal lamina and pericyte detachment from the basal lamina at 6-24 h after LPS injection. The disorganization in the pericyte and basal lamina unit was well correlated with increased microglial activation and increased cerebrovascular permeability in LPS-treated mice. These findings suggest that pericyte detachment and microglial activation may be involved in the mediation of BBB disruption due to inflammatory responses in the damaged brain.

BackgroundIncreased matrix metalloproteinase (MMP)-9 in the plasma and brain is associated with blood-brain barrier (BBB) disruption through proteolytic activity in neuroinflammatory diseases. MMP-9 is present in the brain microvasculature and its vicinity, where brain microvascular endothelial cells (BMECs), pericytes and astrocytes constitute the BBB. Little is known about the cellular source and role of MMP-9 at the BBB. Here, we examined the ability of pericytes to release MMP-9 and migrate in response to inflammatory mediators in comparison with BMECs and astrocytes, using primary cultures isolated from rat brains.MethodsThe culture supernatants were collected from primary cultures of rat brain endothelial cells, pericytes, or astrocytes. MMP-9 activities and levels in the supernatants were measured by gelatin zymography and western blot, respectively. The involvement of signaling molecules including mitogen-activated protein kinases (MAPKs) and phosphoinositide-3-kinase (PI3K)/Akt in the mediation of tumor necrosis factor (TNF)-α-induced MMP-9 release was examined using specific inhibitors. The functional activity of MMP-9 was evaluated by a cell migration assay.ResultsZymographic and western blot analyses demonstrated that TNF-α stimulated pericytes to release MMP-9, and this release was much higher than from BMECs or astrocytes. Other inflammatory mediators [interleukin (IL)-1β, interferon-γ, IL-6 and lipopolysaccharide] failed to induce MMP-9 release from pericytes. TNF-α-induced MMP-9 release from pericytes was found to be mediated by MAPKs and PI3K. Scratch wound healing assay showed that in contrast to BMECs and astrocytes the extent of pericyte migration was significantly increased by TNF-α. This pericyte migration was inhibited by anti-MMP-9 antibody.ConclusionThese findings suggest that pericytes are most sensitive to TNF-α in terms of MMP-9 release, and are the major source of MMP-9 at the BBB. This pericyte-derived MMP-9 initiated cellular migration of pericytes, which might be involved in pericyte loss in the damaged BBB.

The blood-brain barrier (BBB) is formed by brain capillary endothelial cells, astrocytes, pericytes, microglia, and neurons. BBB disruption under pathological conditions such as neurodegenerative disease and inflammation is observed in parallel with microglial activation. To test whether activation of microglia is linked to BBB dysfunction, we evaluated the effect of lipopolysaccharide (LPS) on BBB functions in an in vitro co-culture system with rat brain microvascular endothelial cells (RBEC) and microglia. When LPS was added for 6 h to the abluminal side of RBEC/microglia co-culture at a concentration showing no effects on the RBEC monolayer, transendothelial electrical resistance was decreased and permeability to sodium-fluorescein was increased in RBEC. Immunofluorescence staining for tight junction proteins demonstrated that zonula occludens-1-, claudin-5-, and occludin-like immunoreactivities at the intercellular borders of RBEC were fragmented in the presence of LPS-activated microglia. These functional changes induced by LPS-activated microglia were blocked by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, diphenyleneiodonium chloride. The present findings suggest that LPS activates microglia to induce dysfunction of the BBB by producing reactive oxygen species through NADPH oxidase.

Abstract. The present study was designed to elucidate the involvement of tumor necrosis factor-α (TNF-α ) release from activated microglia in the induction of blood-brain barrier (BBB) dysfunction in an in vitro co-culture system with mouse brain capillary endothelial cells (MBEC4) and microglia. Lipopolysaccharide (LPS)-activated microglia increased the permeability of MBEC4 cells to sodium-fluorescein, and this hyper-permeability was blocked by a neutralizing antibody against TNF-α . LPS stimulated microglia to facilitate TNF-α release. These findings suggested that TNF-α released from activated microglia is attributable to BBB dysfunction.

1) These endothelial cells express specific ion and peptide transporters, which form a transcellular barrier; they also form a paracellular barrier owing to the presence of tight junctions between adjacent endothelial cells.2) The BBB contributes to homeostasis of the central nervous systems (CNS) by restriction of the transport of substances between brain and blood. Reliable in vitro BBB models are required for evaluations of the BBB function, especially the paracellular barrier.Recently, many immortalized brain endothelial cell lines have been established and employed as in vitro BBB models. In this study, we performed comparative studies of the immortalized mouse brain endothelial cell lines bEND.3, bEND.5 and MBEC4 cells, as well as pMBECs. We selected these three mouse cell lines to compare with pMBECs established in many laboratories. MBEC4 cells were established using isolated mouse brain microvascular endothelial cells and showed the highly specialized characteristics of brain microvascular endothelial cells. 8) Tight junction proteins (claudin-5, occludin and ZO-1) are essential for brain endothelial cells to induce and maintain the tightness of tight junctions (the paracellular barrier).9-11) Therefore, we examined the expression levels and distribution of these tight junction proteins and paracellular permeability in the cell lines MBEC4, bEND.3 and bEND.5 cells, and pMBECs. MATERiAlS AND METhODSAnimals ICR mice were obtained from Kyudo (Kumamoto, Japan). Experiments were carried out in compliance with the guidelines stipulated by the Animal Care and Use Committee of Fukuoka University.Isolation and Culture of pMBECs The protocol for isolating pMBECs and growing them was modified from that of Banks et al. 12) Brains from anesthetized 7-8-week-old ICR mice were cleaned of the meninges and homogenized using a hand-held scalpel. Homogenates were digested in a collagenase solution (1 mg/ml collagenase type ii containing 15 µg/ml DNase i) at 37°C for 30 min. Neurons, astrocytes and other cells were removed by centrifugation (1000×g for 20 min) in Dulbecco's modified Eagle's medium (DMEM) containing 20% bovine serum albumin(BSA). The partially purified cell mixture was digested again (1 mg/mL collagenase/dispase containing 6.7 µg/ml DNase i) at 37°C for 30 min. Final purification of endothelial cells was obtained by differential centrifugation on a 33% Percoll gradient (GE Healthcare, Buckinghamshire, U.K.) at 1000×g for 10 min. pMBECs were placed in fibronectin-and collagen IV (both 0.1 mg/mL; Sigma-Aldrich, St. Louis, MO, U.S.A.)-coated culture dishes. Cultures were maintained for 2 d in DMEM/ F12 supplemented with 10% plasma-derived serum, basic fibroblast growth factor (1.5 ng/mL), heparin (100 µg/ml), insulin (5 µg/ml), transferrin (5 µg/ml), sodium selenite (5 ng/ml), gentamicin (50 µg/ml), and puromycin (4 µg/ml) (pMBECs medium i) at 37°C in a humidified atmosphere of 5%CO 2 -95%air. On the third day, pMBECs were cultured in new medium containing all the components of pMBECs medium i except for pur...

1. The present study was aimed at elucidating effects of transforming growth factor-beta (TGF-beta) on blood-brain barrier (BBB) functions with mouse brain capillary endothelial (MBEC4) cells. 2. The permeability coefficients of sodium fluorescein and Evans blue albumin for MBEC4 cells and the cellular accumulation of rhodamine 123 in MBEC4 cells were dose-dependently decreased after a 12-h exposure to TGF-beta1 (0.01-10 ng/mL). 3. The present study demonstrates that TGF-beta lowers the endothelial permeability and enhances the functional activity of P-gp, suggesting that cellular constituents producing TGF-beta in the brain may keep the BBB functioning.