Loss of flow induces leukocyte-mediated MMP/TIMP imbalance in dynamic in vitro blood-brain barrier model: role of pro-inflammatory cytokines

There is substantial evidence linking blood-brain barrier (BBB) failure during cerebral ischemia to matrix metalloproteinases (MMP). BBB function may be affected by loss of shear stress under normoxia/normoglycemia, as during cardiopulmonary bypass procedures. The present study used an in vitro flow-perfused BBB model to analyze the individual contributions of flow, cytokine levels, and circulating blood leukocytes on the release/activity of MMP-9, MMP-2, and their endogenous inhibitors, the tissue inhibitors of MMPs (TIMPs), TIMP-1, and TIMP-2. The presence of circulating blood leukocytes under normoxic/normoglycemic flow cessation/reperfusion significantly increased the luminal levels of MMP-9 and activity of MMP-2, accompanied by partial reduction of TIMP-1, complete reduction of TIMP-2 and increased BBB permeability. These changes were not observed during constant flow with circulating blood leukocytes, or after normoxic/normoglycemic or hypoxic/hypoglycemic flow cessation/reperfusion without circulating blood leukocytes. The addition of anti-IL-6 or anti-TNF- antibody in the lumen before reperfusion suppressed the levels of MMP-9 and activity of MMP-2, had no effect on TIMP-1, and completely restored TIMP-2 and BBB integrity. Injection of TIMP-2 in the lumen before reperfusion prevented the activation of MMP-2 and BBB permeability. These data indicate that blood leukocytes and loss of flow are major factors in the activation of MMP-2, and that cytokine-mediated differential regulation of TIMP-1 and TIMP-2 may contribute significantly to BBB failure. shear stress; inflammation; matrix metalloproteinases     

Nanoparticulate transport of oximes over an in vitro blood-brain barrier model

Background

Due to the use of organophosphates (OP) as pesticides and the availability of OP-type nerve agents, an effective medical treatment for OP poisonings is still a challenging problem. The acute toxicity of an OP poisoning is mainly due to the inhibition of acetylcholinesterase (AChE) in the peripheral and central nervous systems (CNS). This results in an increase in the synaptic concentration of the neurotransmitter acetylcholine, overstimulation of cholinergic receptors and disorder of numerous body functions up to death. The standard treatment of OP poisoning includes a combination of a muscarinic antagonist and an AChE reactivator (oxime). However, these oximes can not cross the blood-brain barrier (BBB) sufficiently. Therefore, new strategies are needed to transport oximes over the BBB.

Methodology/Principal Findings

In this study, we combined different oximes (obidoxime dichloride and two different HI 6 salts, HI 6 dichloride monohydrate and HI 6 dimethanesulfonate) with human serum albumin nanoparticles and could show an oxime transport over an in vitro BBB model. In general, the nanoparticulate transported oximes achieved a better reactivation of OP-inhibited AChE than free oximes.

Conclusions/Significance

With these nanoparticles, for the first time, a tool exists that could enable a transport of oximes over the BBB. This is very important for survival after severe OP intoxication. Therefore, these nanoparticulate formulations are promising formulations for the treatment of the peripheral and the CNS after OP poisoning.     

Permeability properties of a three-cell type in vitro model of blood-brain barrier

We previously found that RBE4.B brain capillary endothelial cells (BCECs) form a layer with blood-brain barrier (BBB) properties if co-cultured with neurons for at least one week. As astrocytes are known to modulate BBB functions, we further set a culture system that included RBE4.B BCECs, neurons and astrocytes. In order to test formation of BBB, we measured the amount of 3H-sucrose able to cross the BCEC layer in this three-cell type model of BBB. Herein we report that both neurons and astrocytes induce a decrease in the permeability of the BCEC layer to sucrose. These effects are synergic as if BCECs are cultured with both neurons and astrocytes for 5 days, permeability to sucrose decreases even more. By Western analysis, we also found that, in addition to the canonical 60 kDa occludin, anti-occludin antibodies recognize a smaller protein of 48 kDa which accumulates during rat brain development. Interestingly this latter protein is present at higher amounts in endothelial cells cultured in the presence of both astrocytes and neurons, that is in those conditions in which sucrose permeation studies indicate formation of BBB.     

Optimization of endothelial cell growth in a murine in vitro blood-brain barrier model

In vitro cell culture models of the blood-brain barrier (BBB) are important tools used to study cellular physiology and brain disease therapeutics. Although the number of model configurations is expanding across neuroscience laboratories, it is not clear that any have been effectively optimized. A sequential screening study to identify optimal primary mouse endothelial cell parameter set points, grown alone and in combination with common model enhancements, including co-culturing with primary mouse or rat astrocytes and addition of biochemical agents in the media, was performed. A range of endothelial cell-seeding densities (1-8 × 10(5) cells/cm(2) ) and astrocyte-seeding densities (2-8 × 10(4) cells/cm(2) ) were studied over seven days in the system, and three distinct media-feeding strategies were compared to optimize biochemical agent exposure time. Implementation of all optimal set points increased transendothelial electrical resistance by over 200% compared to an initial model and established a suitable in vitro model for brain disease application studies. These results demonstrate the importance of optimizing cell culture growth, which is the most important parameter in creating an in vitro BBB model as it directly relates the model to the in vivo arrangement.     

African trypanosome interactions with an in vitro model of the human blood-brain barrier

The neurological manifestations of sleeping sickness in man are attributed to the penetration of the blood-brain barrier (BBB) and invasion of the central nervous system by Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. However, how African trypanosomes cross the BBB remains an unresolved issue. We have examined the traversal of African trypanosomes across the human BBB using an in vitro BBB model system constructed of human brain microvascular endothelial cells (BMECs) grown on Costar Transwell inserts. Human-infective T. b. gambiense strain IL 1852 was found to cross human BMECs far more readily than the animal-infective Trypanosoma brucei brucei strains 427 and TREU 927. Tsetse fly-infective procyclic trypomastigotes did not cross the human BMECs either alone or when coincubated with bloodstreamform T. b. gambiense. After overnight incubation, the integrity of the human BMEC monolayer measured by transendothelial electrical resistance was maintained on the inserts relative to the controls when the endothelial cells were incubated with T. b. brucei. However, decreases in electrical resistance were observed when the BMEC-coated inserts were incubated with T. b. gambiense. Light and electron microscopy studies revealed that T. b. gambiense initially bind at or near intercellular junctions before crossing the BBB paracellularly. This is the first demonstration of paracellular traversal of African trypanosomes across the BBB. Further studies are required to determine the mechanism of BBB traversal by these parasites at the cellular and molecular level.     

Oxidative stress, cytokine/chemokine and disruption of blood-brain barrier in neonate rats after meningitis by Streptococcus agalactiae

We verify the levels of cytokine/chemokine, myeloperoxidase activity, oxidative stress and disruption of BBB in hippocampus and cortex of the neonate Wistar rats after meningitis by S. agalactiae. In the hippocampus the levels were increased of CINC-1 at 6 h and 12 h, IL-1β at 6, 12 and 24 h, IL-6 at 6, 24 and 96 h, IL-10 at 24, 48 and 96 h and TNF-α at 24 h and 96 h. In the cortex the CINC-1 and IL-1β levels were found increased at 6 h. The MPO activity was significantly elevated at 24, 48 and 98 h in hippocampus and at 6, 12, 24, 48 and 96 h in the cortex. The breakdown of BBB started at 12 h.TBARS levels were elevated in the hippocampus at 6, 12, 24, 48, 72 and 96 h and cortex at 72 and 96 h. Protein carbonyls were elevated in the hippocampus and cortex at 6, 24, 48, 72 and 96 h. There was a decrease of SOD activity in hippocampus and in cortex. Catalase activity was elevated in hippocampus at 6 h and in the cortex at 12 and 96 h. Neonatal bacterial infections of the CNS are severe, the interference with the complex network of cytokines/chemokine, other inflammatory mediators and oxidants tend to aggravate the illness and can be involved in the breakdown of the BBB.     

Highly active antiretroviral therapy drug combination induces oxidative stress and mitochondrial dysfunction in immortalized human blood-brain barrier endothelial cells

The era of highly active antiretroviral therapy (HAART) has controlled AIDS and its related disorders considerably; however, the prevalence of HIV-1-associated neurocognitive disorders has been on the rise in the post-HAART era. In view of these developments, we investigated whether a HAART drug combination of 3'-azido-2',3'-deoxythymidine (AZT) and indinavir (IDV) can alter the functionality of the blood-brain barrier (BBB) endothelial cells, thereby exacerbating this condition. The viability of hCMEC/D3 cells (in vitro model of BBB) that were exposed to these drugs was significantly reduced after 72h treatment, in a dose-dependent manner. Reactive oxygen species were highly elevated after the exposure, indicating that mechanisms that induce oxidative stress were involved. Measures of oxidative stress parameters, such as glutathione and malondialdehyde, were altered in the treated groups. Loss of mitochondrial membrane potential, as assessed by fluorescence microscopy and decreased levels of ATP, indicated that cytotoxicity was mediated through mitochondrial dysfunction. Furthermore, AZT+IDV treatment caused apoptosis in endothelial cells, as assessed by the expression of cytochrome c and procaspase-3 proteins. Pretreatment with the thiol antioxidant N-acetylcysteine amide reversed some of the pro-oxidant effects of AZT+IDV. Results from our in vitro studies indicate that the AZT+IDV combination may affect the BBB in HIV-infected individuals treated with HAART drugs.     

Refining in vitro neurotoxicity testing--the development of blood-brain barrier models

The purpose of this paper is to review the current state of development of advanced in vitro blood-brain barrier (BBB) models. The BBB is a special capillary bed that separates the blood from the central nervous system (CNS) parenchyma. Astrocytes maintain the integrity of the BBB, and, without astrocytic contacts, isolated brain capillary endothelial cells in culture lose their barrier characteristics. Therefore, when developing in vitro BBB models, it is important to add astrocytic factors into the culture system. Recently, novel filter techniques and co-culture methods have made it possible to develop models which resemble the in vivo functions of the BBB in an effective way. With a BBB model, kinetic factors can be added into the in vitro batteries used for evaluating the neurotoxic potential of chemicals. The in vitro BBB model also represents a useful tool for the in vitro prediction of the BBB permeability of drugs, and offers the possibility to scan a large number of drugs for their potential to enter the CNS. Cultured monolayers of brain endothelial cell lines or selected epithelial cell lines, combined with astrocyte and neuron cultures, form a novel three-dimensional technique for the screening of neurotoxic compounds.     

Transport of arylsulfatase A across the blood-brain barrier in vitro

Enzyme replacement therapy is an option to treat lysosomal storage diseases caused by functional deficiencies of lysosomal hydrolases as intravenous injection of therapeutic enzymes can correct the catabolic defect within many organ systems. However, beneficial effects on central nervous system manifestations are very limited because the blood-brain barrier (BBB) prevents the transfer of enzyme from the circulation to the brain parenchyma. Preclinical studies in mouse models of metachromatic leukodystrophy, however, showed that arylsulfatase A (ASA) is able to cross the BBB to some extent, thus reducing lysosomal storage in brain microglial cells. The present study aims to investigate the routing of ASA across the BBB and to improve the transfer in vitro using a well established cell culture model consisting of primary porcine brain capillary endothelial cells cultured on Transwell filter inserts. Passive apical-to-basolateral ASA transfer was observed, which was not saturable up to high ASA concentrations. No active transport could be determined. The passive transendothelial transfer was, however, charge-dependent as reduced concentrations of negatively charged monosaccharides in the N-glycans of ASA or the addition of polycations increased basolateral ASA levels. Adsorptive transcytosis is therefore considered to be the major transport pathway. Partial inhibition of the transcellular ASA transfer by mannose 6-phosphate indicated a second route depending on the insulin-like growth factor II/mannose 6-phosphate receptor, MPR300. We conclude that cationization of ASA and an increase of the mannose 6-phosphate content of the enzyme may promote blood-to-brain transfer of ASA, thus leading to an improved therapeutic efficacy of enzyme replacement therapy behind the BBB.     

A cell culture model of the blood-brain barrier

Endothelial cells that make up brain capillaries and constitute the blood-brain barrier become different from peripheral endothelial cells in response to inductive factors found in the nervous system. We have established a cell culture model of the blood-brain barrier by treating brain endothelial cells with a combination of astrocyte-conditioned medium and agents that elevate intracellular cAMP. These cells form high resistance tight junctions and exhibit low rates of paracellular leakage and fluid-phase endocytosis. They also undergo a dramatic structural reorganization as they form tight junctions. Results from these studies suggest modes of manipulating the permeability of the blood-brain barrier, potentially providing the basis for increasing the penetration of drugs into the central nervous system.     

Interaction between flavonoids and the blood-brain barrier: in vitro studies

There is considerable current interest in the neuroprotective effects of flavonoids. This study focuses on the potential for dietary flavonoids, and their known physiologically relevant metabolites, to enter the brain endothelium and cross the blood-brain barrier (BBB) using well-established in vitro models (brain endothelial cell lines and ECV304 monolayers co-cultured with C6 glioma cells). We report that the citrus flavonoids, hesperetin, naringenin and their relevant in vivo metabolites, as well as the dietary anthocyanins and in vivo forms, cyanidin-3-rutinoside and pelargonidin-3-glucoside, are taken up by two brain endothelial cell lines from mouse (b.END5) and rat (RBE4). In both cell types, uptake of hesperetin and naringenin was greatest, increasing significantly with time and as a function of concentration. In support of these observations we report for the first time high apparent permeability (Papp) of the citrus flavonoids, hesperetin and naringenin, across the in vitro BBB model (apical to basolateral) relative to their more polar glucuronidated conjugates, as well as those of epicatechin and its in vivo metabolites, the dietary anthocyanins and to specific phenolic acids derived from colonic biotransformation of flavonoids. The results demonstrate that flavonoids and some metabolites are able to traverse the BBB, and that the potential for permeation is consistent with compound lipophilicity.     

Isoflurane induces endothelial apoptosis of the post-hypoxic blood-brain barrier in a transdifferentiated human umbilical vein endothelial cell model

Isoflurane is a popular volatile anesthetic agent used in humans as well as in experimental animal research. In previous animal studies of the blood-brain barrier (BBB), observations towards an increased permeability after exposure to isoflurane are reported. In this study we investigated the effect of a 2-hour isoflurane exposure on apoptosis of the cerebral endothelium following 24 hours of hypoxia in an in vitro BBB model using astrocyte-conditioned human umbilical vein endothelial cells (AC-HUVECs). Apoptosis of AC-HUVECs was investigated using light microscopy of the native culture for morphological changes, Western blot (WB) analysis of Bax and Bcl-2, and a TUNEL assay. Treatment of AC-HUVECs with isoflurane resulted in severe cellular morphological changes and a significant dose-dependent increase in DNA fragmentation, which was observed during the TUNEL assay analysis. WB analysis confirmed increases in pro-apoptotic Bax levels at 4 hours and 24 hours and decreases in anti-apoptotic Bcl-2 in a dose-dependent manner compared with the control group. These negative effects of isoflurane on the BBB after a hypoxic challenge need to be taken into account not only in experimental stroke research, but possibly also in clinical practice.     

Butyric acid mediated induction of enhanced transendothelial resistance in an in vitro model blood-brain barrier system.

Previously we reported that the co-culture of non-brain vascular endothelial cells with glioma cells leads to the induction of a more differentiated endothelial cell phenotype which exhibits important properties of the blood-brain barrier (BBB). Recognising the potential for improving the model barrier system with agents known to modify the growth and differentiation of cells in culture we examined the effects of four differentiating agents (butyric acid, dexamethasone, retinoic acid, and dimethyl sulfoxide) on barrier function. Of these agents only butyric acid and dexamethasone resulted in an enhancement (depending on the dose used) of transendothelial electrical resistance (barrier function). The greatest effect was observed with butyric acid in a dose-dependent manner and was slow in onset and only occurred in the endothelial/glial cell co-cultures. These data indicate that butyric acid may be a beneficial agent in optimising conditions necessary for induction of BBB properties in in vitro barrier systems.     

Transendothelial permeability changes induced by free radicals in an in vitro model of the blood-brain barrier.

In the present study, we investigated the changes in blood-brain barrier (BBB) permeability following brain endothelial cell exposure to different xenobiotics able to promote free radical generation during their metabolism. Our in vitro BBB model consisted of confluent monolayers of immortalized rat brain capillary endothelial cells (RBE4) grown on collagen-coated filters in the presence of C6 glioma cells grown in the lower compartment. We have recently shown that a range of xenobiotics, including menadione, nitrofurazone, and methylviologen (paraquat) may undergo monoelectronic redox cycling in isolated brain capillaries, giving rise to reactive oxygen species. In this study, addition of 100 microM menadione to the culture medium for 30 min significantly increased the permeability of endothelial cell monolayers to radiolabeled sucrose. The effect on endothelial permeability induced by menadione was dose-dependent and reversible. These permeability changes preceded the onset of cell death, as assessed by the Trypan blue exclusion method. Pre-incubation with superoxide dismutase and catalase blocked changes in sucrose permeability to control levels in a dose-dependent manner, suggesting the involvement of reactive oxygen species in menadione-induced BBB opening.     

Methylated beta-cyclodextrin as P-gp modulators for deliverance of doxorubicin across an in vitro model of blood-brain barrier

Co-incubations of various beta-cyclodextrins and doxorubicin have been evaluated on an in vitro model of blood-brain barrier in order to increase the delivery of this P-gp substrate to the brain. Among these cyclodextrins used, the Rame-beta-cyclodextrin and Crysme-beta-cyclodextrin increased the transport by a factor of 2 and 3.7, respectively. This increase was attributed to the cholesterol extraction property of these cyclodextrins from brain capillary endothelial cells leading to a modulation of the P-gp activity.