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Primary coenzyme Q10 (CoQ10) deficiency is unique among mitochondrial respiratory chain disorders in that it is potentially treatable if high-dose CoQ10 supplements are given in the early stages of the disease.While supplements improve peripheral abnormalities, neurological symptoms are only partially or temporarily ameliorated. The reasons for this refractory response to CoQ10 supplementation are unclear, however, a contributory factor may be the poor transfer of CoQ10 across the blood&ndash, brain barrier (BBB). The aim of this study was to investigate mechanisms of CoQ10 transport across the BBB, using normal and pathophysiological (CoQ10 deficient) cell culture models. The study identifies lipoprotein-associated CoQ10 transcytosis in both directions across the in vitro BBB. Uptake via SR-B1 (Scavenger Receptor) and RAGE (Receptor for Advanced Glycation Endproducts), is matched by efflux via LDLR (Low Density Lipoprotein Receptor) transporters, resulting in no &ldquo, net&rdquo, transport across the BBB. In the CoQ10 deficient model, BBB tight junctions were disrupted and CoQ10 &ldquo, transport to the brain side increased. The addition of anti-oxidants did not improve CoQ10 uptake to the brain side. This study is the first to generate in vitro BBB endothelial cell models of CoQ10 deficiency, and the first to identify lipoprotein-associated uptake and efflux mechanisms regulating CoQ10 distribution across the BBB. The results imply that the uptake of exogenous CoQ10 into the brain might be improved by the administration of LDLR inhibitors, or by interventions to stimulate luminal activity of SR-B1 transporters.

The field of brain drug delivery faces many challenges that hinder development and testing of novel therapies for clinically important central nervous system disorders. Chief among them is how to deliver large biologics across the highly restrictive blood-brain barrier. Non-ionic surfactant vesicles (NISV) have long been used as a drug delivery platform for cutaneous applications and have benefits over comparable liposomes in terms of greater stability, lower cost and suitability for large scale production. Here we describe a glucosamine-coated NISV, for blood-brain barrier GLUT1 targeting, capable of traversing the barrier and delivering active antibody to cells within the brain. In vitro, we show glucosamine vesicle transcytosis across the blood-brain barrier with intact cargo, which is partially dynamin-dependent, but is clathrin-independent and does not associate with sorting endosome marker EEA1. Uptake of vesicles into astrocytes follows a more classical pathway involving dynamin, clathrin, sorting endosomes and Golgi trafficking where the cargo is released intracellularly. In vivo, glucosamine-coated vesicles are superior to uncoated or transferrin-coated vesicles for delivering cargo to the mouse brain. Finally, mice infected with Venezuelan equine encephalitis virus (VEEV) were successfully treated with anti-VEEV monoclonal antibody Hu1A3B-7 delivered in glucosamine-coated vesicles and had improved survival and reduced brain tissue virus levels. An additional benefit was that the treatment also reduced viral load in peripheral tissues. The data generated highlights the huge potential of glucosamine-decorated NISV as a drug delivery platform with wider potential applications.

Elevated serum and cerebrospinal fluid concentrations of S100β, a protein predominantly found in glia, are associated with intracranial injury and neurodegeneration, although concentrations are also influenced by several other factors. The longitudinal association between serum S100β concentrations and brain health in nonpathological aging is unknown. In a large group (baseline N = 593; longitudinal N = 414) of community-dwelling older adults at ages 73 and 76 years, we examined cross-sectional and parallel longitudinal changes between serum S100β and brain MRI parameters: white matter hyperintensities, perivascular space visibility, white matter fractional anisotropy and mean diffusivity (MD), global atrophy, and gray matter volume. Using bivariate change score structural equation models, correcting for age, sex, diabetes, and hypertension, higher S100β was cross-sectionally associated with poorer general fractional anisotropy (r = −0.150, p = 0.001), which was strongest in the anterior thalamic (r = −0.155, p < 0.001) and cingulum bundles (r = −0.111, p = 0.005), and survived false discovery rate correction. Longitudinally, there were no significant associations between changes in brain imaging parameters and S100β after false discovery rate correction. These data provide some weak evidence that S100β may be an informative biomarker of brain white matter aging.

Background: Circulating S100 calcium-binding protein (S100β) is a marker of brain inflammation that has been associated with a range of neurological conditions. To provide insight into the molecular regulation of S100β and its potential causal associations with Alzheimer’s disease, we carried out genome- and epigenome-wide association studies (GWAS/EWAS) of serum S100β levels in older adults and performed Mendelian randomisation with Alzheimer’s disease. Methods: GWAS (N=769, mean age 72.5 years, sd = 0.7) and EWAS (N=722, mean age 72.5 years, sd = 0.7) of S100β levels were performed in participants from the Lothian Birth Cohort 1936. Conditional and joint analysis (COJO) was used to identify independent loci. Expression quantitative trait locus (eQTL) analyses were performed for lead loci that had genome-wide significant associations with S100β. Bidirectional, two-sample Mendelian randomisation was used to test for causal associations between S100β and Alzheimer’s disease. Colocalisation between S100β and Alzheimer’s disease GWAS loci was also examined. Results: We identified 154 SNPs from chromosome 21 that associated (P-8) with S100β protein levels. The lead variant was located in the S100β gene (rs8128872, P=5.0x10-17). We found evidence that two independent causal variants existed for both transcription of S100β and S100β protein levels in our eQTL analyses. No CpG sites were associated with S100β levels at the epigenome-wide significant level (P-8); the lead probe was cg06833709 (P=5.8x10-6), which mapped to the LGI1 gene. There was no evidence of a causal association between S100β levels and Alzheimer’s disease or vice versa and no evidence for colocalisation between S100β and Alzheimer’s disease loci. Conclusions: These data provide insight into the molecular regulators of S100β levels. This context may aid in understanding the role of S100β in brain inflammation and neurological disease.

Perivascular compartments surrounding central nervous system (CNS) vessels have been proposed to serve key roles in facilitating cerebrospinal fluid flow into the brain, CNS waste transfer, and immune cell trafficking. Traditionally, these compartments were identified by electron microscopy with limited molecular characterization. Using cellular markers and knowledge on cellular sources of basement membrane laminins, we here describe molecularly distinct compartments surrounding different vessel types and provide a comprehensive characterization of the arachnoid and pial compartments and their connection to CNS vessels and perivascular pathways. We show that differential expression of plectin, E-cadherin and laminins α1, α2, and α5 distinguishes pial and arachnoid layers at the brain surface, while endothelial and smooth muscle laminins α4 and α5 and smooth muscle actin differentiate between arterioles and venules. Tracer studies reveal that interconnected perivascular compartments exist from arterioles through to veins, potentially providing a route for fluid flow as well as the transport of large and small molecules.

Recent evidence suggests that the ion channel TRPA1 is implicated in lung adenocarcinoma (LUAD), where its role and mechanism of action remain unknown. We have previously established that the membrane receptor FGFR2 drives LUAD progression through aberrant protein–protein interactions mediated via its C-terminal proline-rich motif. Here we report that the N-terminal ankyrin repeats of TRPA1 directly bind to the C-terminal proline-rich motif of FGFR2 inducing the constitutive activation of the receptor, thereby prompting LUAD progression and metastasis. Furthermore, we show that upon metastasis to the brain, TRPA1 gets depleted, an effect triggered by the transfer of TRPA1-targeting exosomal microRNA (miRNA-142-3p) from brain astrocytes to cancer cells. This downregulation, in turn, inhibits TRPA1-mediated activation of FGFR2, hindering the metastatic process. Our study reveals a direct binding event and characterizes the role of TRPA1 ankyrin repeats in regulating FGFR2-driven oncogenic process; a mechanism that is hindered by miRNA-142-3p., TRPA1 has been reported to contribute lung cancer adenocarcinoma (LUAD), but the mechanisms are unclear. Here the authors propose that TRPA1/FGFR2 interaction is functional in LUAD and show that astrocytes oppose brain metastasis by mediating the downregulation of TRPA1 through exosome-delivered miRNA-142-3p.

Brain capillary endothelium mediates the exchange of nutrients between blood and brain parenchyma. This barrier function of the brain capillaries also limits passage of pharmaceuticals from blood to brain, which hinders treatment of several neurological disorders. Receptor-mediated transport has been suggested as a potential pharmaceutical delivery route across the brain endothelium, e.g. reports have shown that the transferrin receptor (TfR) facilitates transcytosis of TfR antibodies, but it is not known whether this recycling receptor itself traffics from apical to basal membrane in the process. Here, we elucidate the endosomal trafficking of the retrograde transported cation-independent mannose-6-phosphate receptor (MPR300) in primary cultures of brain endothelial cells (BECs) of porcine and bovine origin. Receptor expression and localisation of MPR300 in the endo-lysosomal system and trafficking of internalised receptor are analysed. We also demonstrate that MPR300 can undergo bidirectional apical-basal trafficking in primary BECs in co-culture with astrocytes. This is, to our knowledge, the first detailed study of retrograde transported receptor trafficking in BECs, and the study demonstrates that MPR300 can be transported from the luminal to abluminal membrane and reverse. Such trafficking of MPR300 suggests that retrograde transported receptors in general may provide a mechanism for transport of pharmaceuticals into the brain. Brain capillary endothelium mediates the exchange of nutrients between blood and brain parenchyma. This barrier function of the brain capillaries also limits passage of pharmaceuticals from blood to brain, which hinders treatment of several neurological disorders. Receptor-mediated transport has been suggested as a potential pharmaceutical delivery route across the brain endothelium, e.g. reports have shown that the transferrin receptor (TfR) facilitates transcytosis of TfR antibodies, but it is not known whether this recycling receptor itself traffics from apical to basal membrane in the process. Here, we elucidate the endosomal trafficking of the retrograde transported cation-independent mannose-6-phosphate receptor (MPR300) in primary cultures of brain endothelial cells (BECs) of porcine and bovine origin. Receptor expression and localisation of MPR300 in the endo-lysosomal system and trafficking of internalised receptor are analysed. We also demonstrate that MPR300 can undergo bidirectional apical-basal trafficking in primary BECs in co-culture with astrocytes. This is, to our knowledge, the first detailed study of retrograde transported receptor trafficking in BECs, and the study demonstrates that MPR300 can be transported from the luminal to abluminal membrane and reverse. Such trafficking of MPR300 suggests that retrograde transported receptors in general may provide a mechanism for transport of pharmaceuticals into the brain.

Background Brain endothelial cell-based in vitro models are among the most versatile tools in blood–brain barrier research for testing drug penetration to the central nervous system. Transcytosis of large pharmaceuticals across the brain capillary endothelium involves the complex endo-lysosomal system. This system consists of several types of vesicle, such as early, late and recycling endosomes, retromer-positive structures, and lysosomes. Since the endo-lysosomal system in endothelial cell lines of in vitro blood–brain barrier models has not been investigated in detail, our aim was to characterize this system in different models. Methods For the investigation, we have chosen two widely-used models for in vitro drug transport studies: the bEnd.3 mouse and the hCMEC/D3 human brain endothelial cell line. We compared the structures and attributes of their endo-lysosomal system to that of primary porcine brain endothelial cells. Results We detected significant differences in the vesicular network regarding number, morphology, subcellular distribution and lysosomal activity. The retromer-positive vesicles of the primary cells were distinct in many ways from those of the cell lines. However, the cell lines showed higher lysosomal degradation activity than the primary cells. Additionally, the hCMEC/D3 possessed a strikingly unique ratio of recycling endosomes to late endosomes. Conclusions Taken together our data identify differences in the trafficking network of brain endothelial cells, essentially mapping the endo-lysosomal system of in vitro blood–brain barrier models. This knowledge is valuable for planning the optimal route across the blood–brain barrier and advancing drug delivery to the brain. Electronic supplementary material The online version of this article (10.1186/s12987-019-0134-9) contains supplementary material, which is available to authorized users.

Brain fluids are rigidly regulated to provide stable environments for neuronal function, e.g., low K+, Ca2+, and protein to optimise signalling and minimise neurotoxicity. At the same time, neuronal and astroglial waste must be promptly removed. The interstitial fluid (ISF) of the brain tissue and the cerebrospinal fluid (CSF) bathing the CNS are integral to this homeostasis and the idea of a glia-lymph or ‘glymphatic’ system for waste clearance from brain has developed over the last 5 years. This links bulk (convective) flow of CSF into brain along the outside of penetrating arteries, glia-mediated convective transport of fluid and solutes through the brain extracellular space (ECS) involving the aquaporin-4 (AQP4) water channel, and finally delivery of fluid to venules for clearance along peri-venous spaces. However, recent evidence favours important amendments to the ‘glymphatic’ hypothesis, particularly concerning the role of glia and transfer of solutes within the ECS. This review discusses studies which question the role of AQP4 in ISF flow and the lack of evidence for its ability to transport solutes; summarizes attributes of brain ECS that strongly favour the diffusion of small and large molecules without ISF flow; discusses work on hydraulic conductivity and the nature of the extracellular matrix which may impede fluid movement; and reconsiders the roles of the perivascular space (PVS) in CSF–ISF exchange and drainage. We also consider the extent to which CSF–ISF exchange is possible and desirable, the impact of neuropathology on fluid drainage, and why using CSF as a proxy measure of brain components or drug delivery is problematic. We propose that new work and key historical studies both support the concept of a perivascular fluid system, whereby CSF enters the brain via PVS convective flow or dispersion along larger caliber arteries/arterioles, diffusion predominantly regulates CSF/ISF exchange at the level of the neurovascular unit associated with CNS microvessels, and, finally, a mixture of CSF/ISF/waste products is normally cleared along the PVS of venules/veins as well as other pathways; such a system may or may not constitute a true ‘circulation’, but, at the least, suggests a comprehensive re-evaluation of the previously proposed ‘glymphatic’ concepts in favour of a new system better taking into account basic cerebrovascular physiology and fluid transport considerations.

Under a Creative Commons license.-- et al., Carbon nanotubes (CNTs) are a novel nanocarriers with interesting physical and chemical properties. Here we investigate the ability of amino-functionalized multi-walled carbon nanotubes (MWNTs-NH3+) to cross the Blood-Brain Barrier (BBB) in vitro using a co-culture BBB model comprising primary porcine brain endothelial cells (PBEC) and primary rat astrocytes, and in vivo following a systemic administration of radiolabelled f-MWNTs. Transmission Electron microscopy (TEM) confirmed that MWNTs-NH3+ crossed the PBEC monolayer via energy-dependent transcytosis. MWNTs-NH3+ were observed within endocytic vesicles and multi-vesicular bodies after 4 and 24 h. A complete crossing of the in vitro BBB model was observed after 48 h, which was further confirmed by the presence of MWNTs-NH3+ within the astrocytes. MWNT-NH3+ that crossed the PBEC layer was quantitatively assessed using radioactive tracers. A maximum transport of 13.0 ± 1.1% after 72 h was achieved using the co-culture model. f-MWNT exhibited significant brain uptake (1.1 ± 0.3% injected dose/g) at 5 min after intravenous injection in mice, after whole body perfusion with heparinized saline. Capillary depletion confirmed presence of f-MWNT in both brain capillaries and parenchyma fractions. These results could pave the way for use of CNTs as nanocarriers for delivery of drugs and biologics to the brain, after systemic administration., KAJ acknowledges funding from Biotechnology and Biological Sciences Research Council (BB/J008656/1) and Worldwide Cancer Research (12-1054). KAJ, EP and BB acknowledge the EU FP7-ITN Marie-Curie Network programme RADDEL (290023). HK was sponsored by the Atomic Energy Commission of Syria. EP is enrolled in the UAB PhD program. KAJ thanks the members of the EU COST actions TD1004 (Theranostics Imaging and Therapy: An Action to Develop Novel Nanosized Systems for Imaging-Guided Drug Delivery and Biological Processes) for sponsoring HK's research stay at the ICN2 for the electron microscopy studies. ICN2 acknowledges support from the Severo Ochoa Program (MINECO, Grant SEV-2013-0295). Open Access funded by Biotechnology and Biological Sciences Research Council

Key points It is unclear precisely how macromolecules (e.g. endogenous proteins and exogenous immunotherapeutics) access brain tissue from the cerebrospinal fluid (CSF). We show that transport at the brain–CSF interface involves a balance between Fickian diffusion in the extracellular spaces at the brain surface and convective transport in perivascular spaces of cerebral blood vessels. Intrathecally-infused antibodies exhibited size-dependent access to the perivascular spaces and tunica media basement membranes of leptomeningeal arteries. Perivascular access and distribution of full-length IgG could be enhanced by intrathecal co-infusion of hyperosmolar mannitol. Pores or stomata present on CSF-facing leptomeningeal cells ensheathing blood vessels in the subarachnoid space may provide unique entry sites into the perivascular spaces from the CSF. These results illuminate new mechanisms likely to govern antibody trafficking at the brain–CSF interface with relevance for immune surveillance in the healthy brain and insights into the distribution of therapeutic antibodies. Abstract The precise mechanisms governing the central distribution of macromolecules from the cerebrospinal fluid (CSF) to the brain and spinal cord remain poorly understood, despite their importance for physiological processes such as antibody trafficking for central immune surveillance, as well as several ongoing intrathecal clinical trials. In the present study, we clarify how IgG and smaller single-domain antibodies (sdAb) distribute throughout the whole brain in a size-dependent manner after intrathecal infusion in rats using ex vivo fluorescence and in vivo three-dimensional magnetic resonance imaging. Antibody distribution was characterized by diffusion at the brain surface and widespread distribution to deep brain regions along the perivascular spaces of all vessel types, with sdAb accessing a four- to seven-fold greater brain area than IgG. Perivascular transport involved blood vessels of all caliber and putative smooth muscle and astroglial basement membrane compartments. Perivascular access to smooth muscle basement membrane compartments also exhibited size-dependence. Electron microscopy was used to show stomata on leptomeningeal coverings of blood vessels in the subarachnoid space as potential access points allowing substances in the CSF to enter the perivascular space. Osmolyte co-infusion significantly enhanced perivascular access of the larger antibody from the CSF, with intrathecal 0.75 m mannitol increasing the number of perivascular profiles per slice area accessed by IgG by ∼50%. The results of the present study reveal potential distribution mechanisms for endogenous IgG, which is one of the most abundant proteins in the CSF, as well as provide new insights with respect to understanding and improving the drug delivery of macromolecules to the central nervous system via the intrathecal route.

Permanent hearing loss affects more than 5% of the world's population, yet there are no nondevice therapies that can protect or restore hearing. Delivery of therapeutics to the cochlea and vestibular system of the inner ear is complicated by their inaccessible location. Drug delivery to the inner ear via the vasculature is an attractive noninvasive strategy, yet the blood-labyrinth barrier at the luminal surface of inner ear capillaries restricts entry of most blood-borne compounds into inner ear tissues. Here, we compare the blood-labyrinth barrier to the blood-brain barrier, discuss invasive intratympanic and intracochlear drug delivery methods, and evaluate noninvasive strategies for drug delivery to the inner ear.

Most studies of blood-brain barrier (BBB) permeability and transport are conducted at a single pH, but more detailed information can be revealed by using multiple pH values. A pH-dependent biophysical model was applied to the mechanistic analysis of published pH-dependent BBB luminal uptake data from three opioid derivatives in rat: pentazocine (Suzuki et al., 2002a, 2002b), naloxone (Suzuki et al., 2010a), and oxycodone (Okura et al., 2008). Two types of data were processed: in situ brain perfusion (ISBP) and brain uptake index (BUI). The published perfusion data were converted to apparent luminal permeability values, Papp, and analyzed by the pCEL-X program (Yusof et al., 2014), using the pH-dependent Crone-Renkin equation (pH-CRE) to determine the impact of cerebrovascular flow on the Michaelis-Menten transport parameters (Avdeef and Sun, 2011). For oxycodone, the ISBP data had been measured at pH 7.4 and 8.4. The present analysis indicates a 7-fold lower value of the cerebrovascular flow velocity, Fpf, than that expected in the original study. From the pyrilamine-inhibited data, the flow-corrected passive intrinsic permeability value was determined to be P0 = 398 × 10− 6 cm·s− 1. The uptake data indicate that the neutral form of oxycodone is affected by a transporter at pH 8.4. The extent of the cation uptake was less certain from the available data. For pentazocine, the brain uptake by the BUI method had been measured at pH 5.5, 6.5, and 7.4, in a concentration range 0.1–40 mM. Under similar conditions, ISBP data were also available. The pH-CRE determined values of Fpf from both methods were nearly the same, and were smaller than the expected value in the original publication. The transport of the cationic pentazocine was not fully saturated at pH 5.5 at 40 mM. The transport of the neutral species at pH 7.4 appeared to reach saturation at 40 mM pentazocine concentration, but not at 12 mM. In the case of naloxone, a pH-dependent Michaelis-Menten equation (pH-MME) analysis of the data indicated a smooth sigmoidal transition from a higher capacity uptake process affecting cationic naloxone (pH 5.0–7.0) to a lower capacity uptake process affecting the neutral drug (pH 8.0–8.5), with cross-over point near pH 7.4. Evidently, measurements at multiple pH values can reveal important information about both cerebrovascular flow and BBB transport kinetics.

Blood-brain barrier pathology is recognised as a central factor in the development of many neurological disorders, but much less is known about the role of the blood-brain barrier in psychiatric disorders. We review post-mortem, serum-biomarker, CSF-biomarker, and neuroimaging studies that have examined blood-brain barrier structure and function in schizophrenia and related psychoses. We consider how blood-brain barrier dysfunction could relate to glutamatergic and inflammatory abnormalities, which are increasingly understood to play a part in the pathogenesis of psychosis. Mechanisms by which the blood-brain barrier and its associated solute transporters moderate CNS availability of antipsychotic drugs are summarised. We conclude that the complex nature of blood-brain barrier dysfunction in psychosis might be relevant to many aspects of disrupted neuronal and synaptic function, increased permeability to inflammatory molecules, disrupted glutamate homoeostasis, impaired action of antipsychotics, and development of antipsychotic resistance. Future research should address the longitudinal course of blood-brain barrier alterations in psychosis, to determine whether blood-brain barrier dysfunction is a cause or consequence of the pathology associated with the disorder.

The neurons of the central nervous system (CNS) require precise control of their bathing microenvironment for optimal function, and an important element in this control is the blood-brain barrier (BBB). The BBB is formed by the endothelial cells lining the brain microvessels, under the inductive influence of neighbouring cell types within the 'neurovascular unit' (NVU) including astrocytes and pericytes. The endothelium forms the major interface between the blood and the CNS, and by a combination of low passive permeability and presence of specific transport systems, enzymes and receptors regulates molecular and cellular traffic across the barrier layer. A number of methods and models are available for examining BBB permeation in vivo and in vitro, and can give valuable information on the mechanisms by which therapeutic agents and constructs permeate, ways to optimize permeation, and implications for drug discovery, delivery and toxicity. For treating lysosomal storage diseases (LSDs), models can be included that mimic aspects of the disease, including genetically-modified animals, and in vitro models can be used to examine the effects of cells of the NVU on the BBB under pathological conditions. For testing CNS drug delivery, several in vitro models now provide reliable prediction of penetration of drugs including large molecules and artificial constructs with promising potential in treating LSDs. For many of these diseases it is still not clear how best to deliver appropriate drugs to the CNS, and a concerted approach using a variety of models and methods can give critical insights and indicate practical solutions.

This article introduces the special issue on "Blood-Brain Barrier and Epilepsy." We review briefly current understanding of the structure and function of the blood-brain barrier (BBB), including its development and normal physiology, and ways in which it can be affected in pathology. The BBB formed by the endothelium of cerebral blood vessels is one of three main barrier sites protecting the central nervous system (CNS). The barrier is not a rigid structure, but a dynamic interface with a range of interrelated functions, resulting from extremely effective tight junctions, transendothelial transport systems, enzymes, and regulation of leukocyte permeation, which thereby generates the physical, transport, enzymatic, and immune regulatory functions of the BBB. The brain endothelial cells are important components of a "modular" structure, the neurovascular unit (NVU), with several associated cell types and extracellular matrix components. Modern methods have helped in identifying a range of proteins involved in barrier structure and function, and recent studies have revealed important stages, cell types, and signaling pathways important in BBB development. There is a growing list of CNS pathologies showing BBB dysfunction, with strong evidence that this can play a major role in certain disease etiologies. The articles that follow in this issue summarize in more detail reports and discussions of the recent international meeting on "BBB in Neurological Dysfunctions," which took place recently at Ben-Gurion University of the Negev Desert Campus (Beer-Sheva, Israel), focusing on the link between experimental and clinical studies, and the ways in which these lead to improved drug treatments.

International audience; The endothelial cells lining the brain capillaries separate the blood from the brain parenchyma. The endothelial monolayer of the brain capillaries serves both as a crucial interface for exchange of nutrients, gases, and metabolites between blood and brain, and as a barrier for neurotoxic components of plasma and xenobiotics. This "blood-brain barrier" function is a major hindrance for drug uptake into the brain parenchyma. Cell culture models, based on either primary cells or immortalized brain endothelial cell lines, have been developed, in order to facilitate in vitro studies of drug transport to the brain and studies of endothelial cell biology and pathophysiology. In this review, we aim to give an overview of established in vitro blood-brain barrier models with a focus on their validation regarding a set of well-established blood-brain barrier characteristics. As an ideal cell culture model of the blood-brain barrier is yet to be developed, we also aim to give an overview of the advantages and drawbacks of the different models described.

Astrocytic leptin receptors (ObR) can be up-regulated in conditions such as adult-onset obesity. To determine whether the levels and subtypes of astrocytic ObR modulate leptin transport, we co-cultured hCMEC/D3 human brain endothelial cells and C6 astrocytoma cells in the Transwell system, and tested leptin permeation from apical to basolateral chambers. In comparison with hCMEC alone, co-culture of C6 cells reduced the permeability of paracellular markers and leptin. Unexpectedly, ObRb over-expression in C6 cells increased leptin permeation whereas ObRa over-expression showed no effect when compared with the control group of pcDNA-transfected C6 cells. By contrast, the paracellular permeability to the sodium fluorescein control was unchanged by over-expression of ObR subtypes. Leptin remained intact after crossing the monolayer as shown by HPLC and acid precipitation, and this was not affected by C6 cell co-culture or the over-expression of different ObR subtypes. Thus, increased expression of ObRb (and to a lesser extent ObRe) in C6 cells specifically increased the permeation of leptin across the hCMEC monolayer. Consistent with the evidence that the most apparent regulatory changes of ObR during obesity and inflammation occur in astrocytes, the results indicate that astrocytes actively regulate leptin transport across the blood-brain barrier, a mechanism independent of reduction of paracellular permeability.

Neural signalling within the central nervous system (CNS) requires a highly controlled microenvironment. Cells at three key interfaces form barriers between the blood and the CNS: the blood-brain barrier (BBB), blood-CSF barrier and the arachnoid barrier. The BBB at the level of brain microvessel endothelium is the major site of blood-CNS exchange. The structure and function of the BBB is summarised, the physical barrier formed by the endothelial tight junctions, and the transport barrier resulting from membrane transporters and vesicular mechanisms. The roles of associated cells are outlined, especially the endfeet of astrocytic glial cells, and pericytes and microglia. The embryonic development of the BBB, and changes in pathology are described. The BBB is subject to short and long-term regulation, which may be disturbed in pathology. Any programme for drug discovery or delivery, to target or avoid the CNS, needs to consider the special features of the BBB.

There is a paucity of therapies for most neurological disorders--from rare lysosomal storage diseases to major public health concerns such as stroke and Alzheimer's disease. Advances in the targeting of drugs to the CNS are essential for the future success of neurotherapeutics; however, the delivery of many potentially therapeutic and diagnostic compounds to specific areas of the brain is restricted by the blood-brain barrier, the blood-CSF barrier, or other specialised CNS barriers. These brain barriers are now recognised as a major obstacle to the treatment of most brain disorders. The challenge to deliver therapies to the CNS is formidable, and the solution will require concerted international efforts among academia, government, and industry. At a recent meeting of expert panels, essential and high-priority recommendations to propel brain barrier research forward in six topical areas were developed and these recommendations are presented here.

Indexing is a demanding and generally solitary activity requiring particular combinations of mental skills. Drusilla Calvert (indexer) and Joan Abbott (neuroscientist) became interested in the extent to which modern understanding of brain function can give indexers insights into their own mental activities, and possibly help them to work more efficiently and enjoyably. This article summarizes some key points. It is based on a talk given at the Society of Indexers Annual Conference in Durham, July 2006.

The blood-brain barrier, which is formed by the endothelial cells that line cerebral microvessels, has an important role in maintaining a precisely regulated microenvironment for reliable neuronal signalling. At present, there is great interest in the association of brain microvessels, astrocytes and neurons to form functional 'neurovascular units', and recent studies have highlighted the importance of brain endothelial cells in this modular organization. Here, we explore specific interactions between the brain endothelium, astrocytes and neurons that may regulate blood-brain barrier function. An understanding of how these interactions are disturbed in pathological conditions could lead to the development of new protective and restorative therapies.

The brain endothelium forms the blood–brain barrier (BBB) and is the chief site regulating molecular traffic between blood and brain. The ‘barrier phenotype’ includes tight junctions restricting paracellular flux and a range of transport mechanisms controlling transcellular flux. The barrier is induced by cell types associated with the microvessels and is subject to regulation. The composition of the BBB membranes will influence the permeability of lipid-soluble compounds. Specific transporter systems for solute uptake are present on both the apical (luminal) and basal (abluminal) membranes and efflux transporters of broader specificity are also present. Certain larger molecules may cross via transcytotic vesicular mechanisms. Potential drug molecules designed to enter the brain may use or interact with one or more of these routes—better understanding is needed before it will be possible to establish the optimal means of delivery for specific compounds and to design or modify them accordingly. Many lipid-soluble drugs are substrates for efflux transporters, making it difficult to guarantee CNS delivery or calculate free concentration in the brain interstitial fluid (ISF). The dynamics of the flowing ISF also complicate modelling and prediction of CNS pharmacokinetics. Finally, many disease states involve BBB dysfunction, which needs to be taken into account in designing appropriate therapies.

Object. Drug delivery across the blood—brain barrier remains a significant challenge. Based on earlier findings, the authors hypothesized that parasympathetic innervation of the brain vasculature could be used to augment drug delivery to the brain. Methods. Using a craniotomy—cerebrospinal fluid superfusate paradigm in rats with an intravenous injection of tracer the authors demonstrated that stimulation of the postganglionic parasympathetic fibers of the sphenopalatine ganglion (SPG) increased the concentration of fluorescein isothiocyanate—dextran (4–250 kD) in the superfusate by two- to sixfold. A histological examination indicated the presence of dextran in the parenchyma. In another experiment the amount of Evans blue dye in the brain following SPG activation was similarly significantly elevated. The chemotherapeutic agents anti-HER2 monoclonal antibody and etoposide were also delivered to the brain and reached therapeutic concentrations. Brain homeostasis was not disturbed by this procedure; a measurement of nicotinamide adenine dinucleotide reduction did not show a decrease in the tissue metabolic state and brain water content did not increase significantly. Conclusions. Sphenopalatine ganglion activation demonstrates a promising potential for clinical use in the delivery of small and large molecules to the brain.

The blood-brain barrier (BBB) efflux transport of [(14)C] adenosine was studied using the brain efflux index (BEI) technique. BEI increased linearly over the first 2 min after injection, with deviation from linearity thereafter; 90.12 +/- 1.5% of the injected [(14)C] radioactivity remained within the brain after 20 min. The remaining tracer appears to be mainly intracellular, trapped by phosphorylation, as an almost linear increase of BEI over 20 min was observed after intracerebral injection of [(14)C] adenosine together with 5-iodo tubercidin. The BBB efflux clearance of [(14)C] radioactivity was estimated to be 27.62 +/- 5.2 micro L/min/g, almost threefold higher than the BBB influx clearance estimated by the brain uptake index technique. High-performance liquid chromatography (HPLC) analysis of blood plasma collected from the jugular vein after the intracerebral injection revealed metabolic breakdown of [(14)C] adenosine into nucleobases. The BBB efflux transport was saturable with apparent K(m) = 13.22 +/- 1.75 micro m and V(max) = 621.07 +/- 71.22 pmole/min/g, which indicated that BBB efflux in vivo is 6.2-12p mole/min/g, negligible when compared to the reported rate of adenosine uptake into neurones/glia. However, these kinetic parameters also suggest that under conditions of elevated ISF adenosine in hypoxia/ischaemia, BBB efflux transport could increase up to 25% of the uptake into neurones/glia and become an important mechanism to oppose the rise in ISF concentration. HPLC-fluorometry detected 93.6 +/- 5.25 nm of adenosine in rat plasma, which is 17- to 220-fold lower than the reported K(m) of adenosine BBB influx in rat. Together with the observed rapid degradation inside endothelial cells, this indicated negligible BBB influx of intact adenosine under resting conditions. Cross-inhibition studies showed that unlabelled inosine, adenine and hypoxanthine caused a decrease in BBB efflux of [(14)C] radioactivity in a concentration-dependent manner, with K(i) of 16.7 +/- 4.88, 65.1 +/- 14.1 and 71.1 +/- 16.9 micro m, respectively. This could be due to either competition of unlabelled molecules with [(14)C] adenosine or competition with its metabolites hypoxanthine and adenine for the same transport sites.

A number of nucleoside analogues, consisting of antiviral compounds and agents designed as adenosine A1 receptor agonists, were examined for nucleoside transporter affinity using an in vitro model of the blood-brain barrier (BBB), the rat brain endothelial cell line, RBE4. Structure-activity relationships (SAR) were also performed to identify the key structural requirements for transporter recognition and the suitability of these systems for carrier-mediated strategies to deliver therapeutics across the BBB. Adenosine receptor agonists did not show transport affinity for concentrative nucleoside carriers, but exhibited affinity for equilibrative systems (Ki=10.8-97.9 microM) within the range of Kms for natural substrates. However, none of the antiviral compounds tested in this study showed affinity for either class of nucleoside transporter. SAR studies suggest that the hydroxyl group located at the 3'-position of the ribose moiety is an essential requirement for transporter recognition. This may explain the inability of nucleoside derived anti-viral compounds to use these systems despite the significant structural homology with naturally occurring nucleosides. Sites have also been identified which accommodate structural additions with retention of carrier affinity, suggesting that compounds which fail to penetrate the BBB could be attached to these sites for carrier-mediated delivery using a prodrug strategy.

Understanding mechanisms associated with flavonoid neuroprotection is complicated by the lack of information on their ability to enter the CNS. This study examined naringenin and quercetin permeability across the blood-brain barrier (BBB), using in vitro (ECV304/C6 coculture) and in situ (rat) models. We report measurable permeabilities (P(app)) for both flavonoids across the in vitro BBB model, consistent with their lipophilicity. Both flavonoids showed measurable in situ BBB permeability. The rates of uptake (K(in)) into the right cerebral hemisphere were 0.145 and 0.019 ml min(-1) g(-1) for naringenin and quercetin, respectively. Quercetin K(in) was comparable to that of colchicine (0.006 ml min(-1) g(-1)), a substrate for P-glycoprotein (P-gp). Preadministration of the P-gp inhibitor PSC833 or GF120918 (10 mg/kg body wt) significantly increased colchicine K(in), but only GF120918 (able to inhibit breast cancer resistance protein, BCRP) affected K(in) for quercetin. Naringenin K(in) was not affected. The influence of efflux transporters on flavonoid permeability at the BBB was further studied using MDCK-MDR1 and immortalized rat brain endothelial cells (RBE4). Colchicine, quercetin, and naringenin all showed measurable accumulation (distribution volume, V(d) (microl/mg protein)) in both cell types. The V(d) for colchicine increased significantly in both cell lines following coincubation with either PSC833 (25 microM) or GF120918 (25 microM). Both inhibitors also caused an increase in naringenin V(d); by contrast only GF120918 coincubation significantly increased quercetin V(d). In conclusion, the results demonstrate that flavonoids are able to traverse the BBB in vivo. However, the permeability of certain flavonoids in vivo is influenced by their lipophilicity and interactions with efflux transporters.

Long term treatment with antiepileptic drugs (AEDs) is the standard therapeutic approach to eradicate seizures. However, a small but significant number of patients fail AED treatment. Intrinsic drug resistance may depend on two main and not necessarily mutually exclusive mechanisms: 1) Loss of pharmacological target (e.g., GABAA receptors); 2) poor penetration of the drug into the central nervous system (CNS). The latter is due to the action of multiple drug resistance proteins capable of active CNS extrusion of drugs. These include MDR1 (P-glycoprotein, PgP), the multidrug resistance related proteins MRP1-5, and lung-resistance protein (LRP). Overexpression of MDR1 occurs in human epileptic brain. It has therefore been proposed that MDR1/PgP may contribute to multiple drug resistance in epilepsy. In addition to MDR1/PgP, other genes such as MRP2, MRP5, and human cisplatin resistance-associated protein are also overexpressed in drug-resistant epilepsy. In normal brain tissue MDR1/PgP is expressed almost exclusively by endothelial cells (EC), while in epileptic cortex both EC and perivascular astrocytes express MDR1/PgP. The underlying causes for tissue differences may be genomic (i.e., at the DNA level), or MDR1/PgP could be induced by seizures, previous drug treatment, or a combination of the above. We will present evidence showing that expression of multiple drug resistance genes in epilepsy is a complex phenomenon and that glial cells are involved. This second line of defense for xenobiotics may have profound implications for the pharmacokinetic properties of antiepileptic drugs and their capacity to reach neuronal targets.

Multiple nucleoside transport systems exist in the body yet the subtypes functional at the blood-brain barrier (BBB) have not been fully investigated. We have employed RBE4 immortalised rat brain endothelial cells to functionally identify the carrier subtypes involved in nucleoside transfer between blood and brain. Uptake in RBE4 cells was partially sodium dependent, indicating the presence of both equilibrative and concentrative systems. Uptake of adenosine via equilibrative transporters was sensitive to nitro-benzylmercaptopurine riboside, which showed biphasic inhibition. Uptake of [3H]-adenosine via concentrative transporters was studied using the subtype-specific inhibitors thymidine (cit), formycin-B (cif) and tubercidin (cib) and was significantly reduced by thymidine and formycin-B but not by tubercidin. This study suggests that nucleoside transport at the in situ BBB may be mediated by ei and es equilibrative transporters and by cit and cif concentrative transporters.

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.

The blood-brain barrier (BBB) was modelled in this study using ECV304 cells in co-culture with rat C6 glioma cells, which resulted in elevated transendothelial electrical resistance (TEER). The inflammatory mediator bradykinin (1 microM) was studied and found to induce a fall in TEER; the link between this change and intracellular free calcium concentration ([Ca(2+)](i)) was then examined. 1 microM bradykinin produced a peak-plateau increase in [Ca(2+)](i). The peak showed desensitization and was dose dependent (over 0.1 nM to 1 microM). The [Ca(2+)](i) increase was blocked by the B(2) antagonist HOE 140 (1 microM) without effect from a B(1) agonist and antagonist. The plateau response was abolished in Ca(2+)-free solution containing 2 mM EDTA, and also by the Ca(2+) channel blockers lanthanum, La(3+) (10 microM), and SKF 96365 (100 microM). The store Ca(2+)ATPase inhibitor thapsigargin (1 microM) abolished the peak response. The putative phospholipase C inhibitors, U73122 (20 microM) and ETH-18-OCH(3) (100 microM), unexpectedly increased [Ca(2+)](i); after their application, bradykinin was ineffective. Agents without effect on Ca(2+) responses to bradykinin included the phospholipase A(2) (PLA(2)) inhibitor aristolochic acid (0.5 mM), cyclooxygenase inhibitor indomethacin (100 microM), 5-lipoxygenase inhibitor nordihydroguaiaretic acid, NDGA (100 microM), calphostin C (0.5 microM), L-NAME (1 mM) and nifedipine (10 microM). The fall in TEER from bradykinin was blocked by HOE 140, U73122 and thapsigargin combined with La(3+), and also by aristolochic acid and NDGA, but not indomethacin, calphostin C or L-NAME. U73122 increased TEER while ETH-18-OCH(3) reduced it. Thus bradykinin reduced TEER through B(2) receptor-linked release of Ca(2+) from thapsigargin-sensitive stores, leading to activation of PLA(2) and metabolism of arachidonic acid by 5-lipoxygenase.

The restrictive nature of the blood-brain barrier means that cellular machinery must be in place to deliver macromolecules to the brain. This is achieved by transcytosis which is more complex than initially supposed, both in terms of structure and regulation. Brain endothelial cells have relatively few pinocytotic vesicles compared to peripheral endothelia but can still deliver macromolecules via one of the three main types of vesicles: the most numerous clathrin-coated vesicles containing adaptor protein complex-2, the smaller caveolae formed from lipid raft domains of the plasma membrane, and the large fluid engulfing macropinocytotic vesicles. Both clathrin-coated vesicles and, to a lesser extent caveolae, endocytose plasma membrane receptors and their specific ligands which include insulin, transferrin, and lipoproteins. This receptor-mediated transcytosis (RMT) delivers the ligands to the brain and enables their receptors to be recycled back to the plasma membrane. However, once endocytosed, the ligands and/or receptors must be directed toward the correct plasma membrane and avoid degradation. How this is achieved has not been well studied although there is an important role for Rab GTPases in targeting vesicles to their correct location and enabling exocytosis. In this chapter, we discuss what is known about regulation of transcytosis in related cells such as the MDCK cell line and where are the gaps in our knowledge of brain endothelial transcytotic regulation. We discuss how RMT has been exploited to deliver therapeutic drugs to the brain and the importance of further investigation in this area to improve drug delivery.

Cell culture models of the blood-brain barrier (BBB) are useful tools to study the functionality of the BBB in health and disease. Several good in vitro BBB models are available from different species. However, most brain endothelial cells lose some of their in vivo BBB phenotype in culture. Porcine brain endothelial cells (PBECs) tend to retain most of their in vivo BBB characteristics and usually give higher transendothelial electrical resistance (TEER, representing functional well-developed tight junctions) compared to brain endothelial cells from other species. The protocol described in this unit gives detailed instructions for isolation and culture of PBECs from fresh porcine brains. This porcine BBB model generates high TEER without the need for co-culture with astrocytes. However, astrocyte-derived factors can be introduced to the system through the use of astrocyte-conditioned medium or co-culture with astrocytes, which may be necessary for further enhancing the BBB phenotype for certain complex studies.

The properties of the blood-brain barrier (BBB) are important considerations when designing drugs to target or avoid the brain. In vivo models provide some of the most reliable measurements of BBB drug permeability, whereas several new cell-based in vitro technologies give valuable mechanistic insights. Higher throughout methods (cell- and non-cell-based; in silico modelling) can help in prediction of permeation, but a combination of techniques is still required to cover the range of BBB drug entry and efflux mechanisms.

In vitro blood–brain barrier (BBB) models from primary brain endothelial cells can closely resemble the in vivo BBB, offering valuable models to assay BBB functions and to screen potential central nervous system drugs. We have recently developed an in vitro BBB model using primary porcine brain endothelial cells. The model shows expression of tight junction proteins and high transendothelial electrical resistance, evidence for a restrictive paracellular pathway. Validation studies using small drug-like compounds demonstrated functional uptake and efflux transporters, showing the suitability of the model to assay drug permeability. However, one limitation of in vitro model permeability measurement is the presence of the aqueous boundary layer (ABL) resulting from inefficient stirring during the permeability assay. The ABL can be a rate-limiting step in permeation, particularly for lipophilic compounds, causing underestimation of the permeability. If the ABL effect is ignored, the permeability measured in vitro will not reflect the permeability in vivo. To address the issue, we explored the combination of in vitro permeability measurement using our porcine model with the pKaFLUX method in pCEL-X software to correct for the ABL effect and allow a detailed analysis of in vitro (transendothelial) permeability data, Papp. Published Papp using porcine models generated by our group and other groups are also analyzed. From the Papp, intrinsic transcellular permeability (P0) is derived by simultaneous refinement using a weighted nonlinear regression, taking into account permeability through the ABL, paracellular permeability and filter restrictions on permeation. The in vitro P0 derived for 22 compounds (35 measurements) showed good correlation with P0 derived from in situ brain perfusion data (r2=0.61). The analysis also gave evidence for carrier-mediated uptake of naloxone, propranolol and vinblastine. The combination of the in vitro porcine model and the software analysis provides a useful tool to better predict BBB permeability in vivo and gain better mechanistic information about BBB permeation.

In earlier studies insects were proposed as suitable models for vertebrate blood-brain barrier (BBB) permeability prediction and useful in early drug discovery. Here we provide transcriptome and functional data demonstrating the presence of a P-glycoprotein (Pgp) efflux transporter in the brain barrier of the desert locust (Schistocerca gregaria). In an in vivo study on the locust, we found an increased uptake of the two well-known Pgp substrates, rhodamine 123 and loperamide after co-administration with the Pgp inhibitors cyclosporine A or verapamil. Furthermore, ex vivo studies on isolated locust brains demonstrated differences in permeation of high and low permeability compounds. The vertebrate Pgp inhibitor verapamil did not affect the uptake of passively diffusing compounds but significantly increased the brain uptake of Pgp substrates in the ex vivo model. In addition, studies at 2°C and 30°C showed differences in brain uptake between Pgp-effluxed and passively diffusing compounds. The transcriptome data show a high degree of sequence identity of the locust Pgp transporter protein sequences to the human Pgp sequence (37%), as well as the presence of conserved domains. As in vertebrates, the locust brain-barrier function is morphologically confined to one specific cell layer and by using a whole-brain ex vivo drug exposure technique our locust model may retain the major cues that maintain and modulate the physiological function of the brain barrier. We show that the locust model has the potential to act as a robust and convenient model for assessing BBB permeability in early drug discovery. ispartof: Pharmacology Research & Perspectives vol:2 issue:4 ispartof: location:United States status: published

This chapter reviews the history and modern applications of isolated preparations of the three main CNS barrier layers and cell culture preparations derived from them. In vitro models give valuable mechanistic information but also provide useful assay systems for drug discovery and delivery programmes. However, it is important to take into account practical issues including species differences and the degree to which the differentiated state of the in vivo barrier is retained. The range of models available is reviewed, with a critical evaluation of their strengths and weaknesses, and guidance in selecting and optimizing a suitable model for particular applications. New understanding of the unstirred water layers and paracellular leak pathway in in vitro preparations gives greater insights into the “intrinsic permeability” of the membrane, and a variety of techniques permit characterization of the transport systems and enzymes contributing to barrier function. Increasingly, aspects of CNS pathology are being modelled in cell culture, aiding the optimization of drug delivery regimes in pathological conditions.

This chapter covers the three main barrier layers separating blood and the central nervous system (CNS): the endothelium of the brain vasculature, the epithelium of the choroid plexus secreting cerebrospinal fluid (CSF) into the ventricles and the arachnoid epithelium forming the middle layer of the meninges on the brain surface. There are three key barrier features at each site that control the composition of brain fluids and regulate CNS drug permeation: (1) physical barriers result from features of the cell membranes and of the tight junctions restricting the paracellular pathway through intercellular clefts; (2) transport barriers result from membrane transporters mediating solute uptake and efflux, together with vesicular mechanisms mediating transcytosis of larger molecules such as peptides and proteins and (3) enzymatic barriers result from cell surface and intracellular enzymes that can modify molecules in transit. Brain fluids (CSF and brain interstitial fluid) are secreted, flow through particular routes and then drain back into the venous system; this fluid turnover aids central homeostasis and also affects CNS drug concentration. Several CNS pathologies involve changes in the barrier layers and the fluid systems. Many of these aspects of physiology and pathology have implications for drug delivery.

1. Unlike some interfaces between the blood and the nervous system (e.g., nerve perineurium), the brain endothelium forming the blood–brain barrier can be modulated by a range of inflammatory mediators. The mechanisms underlying this modulation are reviewed, and the implications for therapy of the brain discussed.

DESPITE MAJOR ADVANCES in neuroscience, potential therapeutic options for the treatment of central nervous system diseases often cannot be optimized secondary to the presence of the blood-brain barrier (BBB). During the next decade of inquiry, it is crucial that basic science and clinical research that is focused on overcoming the BBB, to optimize delivery to the central nervous system, be identified and supported as a priority topic. For this reason, the third international Cerebrovascular Biology and Blood-Brain Barrier Conference was convened in March 1998 in Gleneden Beach, OR. This meeting brought together basic science and clinical researchers from around the world to analyze BBB function and to discuss delivery of effective agents to the central nervous system for treatment of brain disease. This report summarizes the information presented at the meeting and the discussions that ensued. The current state of knowledge, obstacles to further understanding the BBB, and research priorities are identified.

The role of Na+–Ca2+ exchange in the regulation of the cytosolic free Ca2+ concentration ([Ca2+]i) was studied in primary cultured rat brain capillary endothelial cells. [Ca2+]i was measured by digital fluorescence imaging in cells loaded with fura-2. ATP (100 μm) applied for a short period of time (6 s) caused a rise in [Ca2+]i from 127 ± 3 (n = 290) to 797 ± 25 nm, which then declined to the resting level, with a t½ (time required for [Ca2+]i to decline to half of peak [Ca2+]i) of 5.4 ± 0.09 s. This effect was independent of external Ca2+ and could be abolished by previously discharging the Ca2+ pool of the endoplasmic reticulum with thapsigargin (1 μm). Application of thapsigargin (1 μm) or cyclopiazonic acid (10 μm) to inhibit the Ca2+-ATPase of the endoplasmic reticulum 6 s prior to ATP application did not influence the peak [Ca2+]i but greatly reduced the rate of decline of [Ca2+]i, with t½ values of 15 ± 1.6 and 23 ± 3 s, respectively. In the absence of external Na+ (Na+ replaced by Li+ or N-methylglucamine) the basal [Ca2+]i was slightly elevated (152 ± 6 nm) and the restoration of [Ca2+]i after the ATP stimulation was significantly slower (t½, 7.3 ± 0.46 s in Li+ medium, 8.12 ± 0.4 s in N-methylglucamine medium). The external Na+-dependent component of the [Ca2+]i sequestration was also demonstrated in cells stimulated by ATP subsequent to addition of cyclopiazonic acid; in a Na+-free medium [Ca2+]i remained at the peak level in 88% of the cells after stimulation with ATP. Addition of monensin (10 μm) in the presence of external Na+ increased the resting [Ca2+]i to 222 ± 9 nm over ∼1 min and subsequent removal of extracellular sodium resulted in a further increase in [Ca2+]i to a peak of 328 ± 11 nm, which was entirely dependent on external Ca2+. These findings indicate that a functional Na+–Ca2+ exchanger is present at the blood-brain barrier, which plays a significant role in shaping the stimulation-evoked [Ca2+]i signal and is able to work in reverse mode under pharmacological conditions. Intracellular calcium is an important signalling mechanism in endothelial cells including the brain microvessel endothelium, leading to the generation and release of vasoactive compounds such as nitric oxide (Weih et al. 1998), prostacyclin (Wiemer et al. 1994) and endothelin (Bacic et al. 1992). Several substances are able to activate the endothelial cells of brain microvessels that form the blood-brain barrier (BBB) leading to a rapid rise in [Ca2+]i (Abbott, 1998a). These include bradykinin (Revest et al. 1991; Wiemer et al. 1994), acetylcholine (Luiten et al. 1996), angiotensin II (Stanimirovic et al. 1996), ADP (Frelin et al. 1993), ATP and purine analogues (Vigne et al. 1994; Nobles et al. 1995; Albert et al. 1997), vasopressin (Bacic et al. 1992), histamine (Revest et al. 1991), serotonin (Olesen, 1989), bombesin (Vigne et al. 1995) and endothelin (Vigne et al. 1990; Kawai et al. 1997), as well as mechanical stimuli (Popp et al. 1992). In addition to establishing a link between stimulation and secretion, calcium signalling leads to changes in cell morphology and BBB permeability through mechanisms that may include activation of myosin light-chain kinase and other cytoskeletal components (Abbott & Revest, 1991; Fleming et al. 1995; Abbott & Romero, 1996; Bartus et al. 1996; Albert et al. 1997; Boarder & Hourani, 1998; Denker & Nigam, 1998). The spatial and temporal characteristics of the [Ca2+]i signal are likely to play an important role in the regulation of Ca2+-dependent intracellular processes, and therefore Ca2+ transporters, which contribute to the shaping of the stimulation-evoked [Ca2+]i signal, are likely to have functional importance. The Na+-Ca2+ exchanger present in the plasma membrane has been shown to play a crucial role in regulating [Ca2+]i in several cell types including cardiac myocytes (Allen et al. 1989), smooth muscle (Batlle et al. 1991) and neurons (Reuter & Porzig, 1995; see also Blaustein, 1984). There is little information available on the Na+-Ca2+ exchange mechanism in endothelial cells. No evidence for the existence of the Na+-Ca2+ exchanger has been found in studies performed on endothelial cells cultured from bovine aorta (Schilling et al. 1988), atria (Laskey et al. 1990) or pulmonary artery (Cannell & Sage, 1989). However, it has been reported that removal of extracellular Na+ or loading the cells with Na+ resulted in an increased [Ca2+]i in cultured endothelial cells from pulmonary artery (Sage et al. 1991) and in intact endothelial cells prepared from cardiac valve (Li & Van Breemen, 1995), thus the existence of the Na+-Ca2+ exchanger has been suggested in these cells. Supporting evidence comes from an immunofluorescence study detecting cross-reaction of antibodies raised against cardiac Na+-Ca2+ exchanger in cultured endothelial cells derived from bovine aorta (Juhaszova et al. 1994). Functional studies on cultured bovine pulmonary artery endothelial cells showed that although external Na+ had a small effect on [Ca2+]i, this appeared not to have primary importance in regulating resting [Ca2+]i (Sage et al. 1991). However, in intact endothelium from cardiac valve, robust changes in the resting [Ca2+]i were detected upon manipulating [Na+]i (Li & van Breemen, 1995). It is unclear whether this difference derives from altered characteristics of the exchanger in cultured cells as compared with that of in situ endothelial cells, or whether the distribution and the physiological role of the Na+-Ca2+ exchanger is different in endothelial cells of different origin. It is not yet known whether the Na+-Ca2+ exchange mechanism exists in brain capillary endothelial cells. The brain endothelium is distinct from that of most other tissues in showing upregulation of a number of features related to its barrier-forming properties in situ. These include complex and extremely tight ‘tight junctions’ (zonulae occludentes) (Wolburg et al. 1994), and a range of specific carrier systems important in regulating transendothelial transport, including carriers mediating brain entry of glucose, amino acids and nucleosides, and efflux carriers such as P-glycoprotein protecting the brain from xenobiotics in the blood (Abbott & Romero, 1996). There is evidence that the permeability of the paracellular (tight junctional) pathway can be modulated by inflammatory mediators, and in some cases a receptor-mediated elevation of [Ca2+]i is implicated in barrier opening (Abbott & Revest 1991; Abbott & Romero, 1996; Abbott, 1998a,b). The BBB phenotype is induced in brain endothelium in vivo by chemical factors secreted by cells of the nervous system, including the endfeet of astrocytic glia that surround the endothelium. Many of the phenotypic features of BBB endothelium are retained in primary cultured brain endothelial cells, although some are progressively lost during subsequent passage (Abbott et al. 1992; Wolburg et al. 1994). In the present study we investigated whether a functional Na+-Ca2+ exchanger is present in primary cultured brain capillary endothelial cells. In addition to studies on the resting [Ca2+]i, the regulation of the stimulation-evoked [Ca2+]i signal was also addressed. ATP was used to stimulate cells via purinergic (P2) receptors present in the plasma membrane of rat brain capillary endothelial cells (RBCECs) (Nobles et al. 1995). We report here the existence of a Na+-Ca2+ exchange mechanism in cultured RBCECs which has only a small effect on the resting [Ca2+]i but significantly contributes to the sequestration of the stimulation-evoked [Ca2+]i rise.