T-Lymphocytes Traffic into the Brain across the Blood-CSF Barrier: Evidence Using a Reconstituted Choroid Plexus Epithelium

An emerging concept of normal brain immune surveillance proposes that recently and moderately activated central memory T lymphocytes enter the central nervous system (CNS) directly into the cerebrospinal fluid (CSF) via the choroid plexus. Within the CSF space, T cells inspect the CNS environment for cognate antigens. This gate of entry into the CNS could also prevail at the initial stage of neuroinflammatory processes. To actually demonstrate T cell migration across the choroidal epithelium forming the blood-CSF barrier, an in vitro model of the rat blood-CSF barrier was established in an “inverse” configuration that enables cell transmigration studies in the basolateral to apical, i.e. blood/stroma to CSF direction. Structural barrier features were evaluated by immunocytochemical analysis of tight junction proteins, functional barrier properties were assessed by measuring the monolayer permeability to sucrose and the active efflux transport of organic anions. The migratory behaviour of activated T cells across the choroidal epithelium was analysed in the presence and absence of chemokines. The migration pathway was examined by confocal microscopy. The inverse rat BCSFB model reproduces the continuous distribution of tight junction proteins at cell margins, the restricted paracellular permeability, and polarized active transport mechanisms, which all contribute to the barrier phenotype in vivo. Using this model, we present experimental evidence of T cell migration across the choroidal epithelium. Cell migration appears to occur via a paracellular route without disrupting the restrictive barrier properties of the epithelial interface. Apical chemokine addition strongly stimulates T cell migration across the choroidal epithelium. The present data provide evidence for the controlled migration of T cells across the blood-CSF barrier into brain. They further indicate that this recruitment route is sensitive to CSF-borne chemokines, extending the relevance of this migration pathway to neuroinflammatory and neuroinfectious disorders which are typified by elevated chemokine levels in CSF.     

Laminins and their receptors in the CNS

Laminin, an extracellular matrix protein, is widely expressed in the central nervous system (CNS). By interacting with integrin and non‐integrin receptors, laminin exerts a large variety of important functions in the CNS in both physiological and pathological conditions. Due to the existence of many laminin isoforms and their differential expression in various cell types in the CNS, the exact functions of each individual laminin molecule in CNS development and homeostasis remain largely unclear. In this review, we first briefly introduce the structure and biochemistry of laminins and their receptors. Next, the dynamic expression of laminins and their receptors in the CNS during both development and in adulthood is summarized in a cell‐type‐specific manner, which allows appreciation of their functional redundancy/compensation. Furthermore, we discuss the biological functions of laminins and their receptors in CNS development, blood–brain barrier (BBB) maintenance, neurodegeneration, stroke, and neuroinflammation. Last, key challenges and potential future research directions are summarized and discussed. Our goals are to provide a synthetic review to stimulate future studies and promote the formation of new ideas/hypotheses and new lines of research in this field.     

The blood-CSF barrier explained: when development is not immaturity

It is often suggested that during development the brain barriers are immature. This argument stems from teleological interpretations and experimental observations of the high protein concentrations in fetal cerebrospinal fluid (CSF) and decreases in apparent permeability of passive markers during development. We argue that the developmental blood-CSF barrier restricts the passage of lipid-insoluble molecules by the same mechanism as in the adult (tight junctions) rendering the paracellular pathway an unlikely route of entry. Instead, we suggest that both protein and passive markers are transferred across the epithelium through a transcellular route. We propose that changes in volume of distribution can largely explain the decrease in apparent permeability for passive markers and that developmentally regulated cellular transfer explains changes in CSF protein concentrations. The blood-CSF tight junctions are functionally mature from very early in development, and it appears that transfer from blood into embryonic brain occurs predominately via CSF rather than the vasculature.     

Proteome analysis of chick embryonic cerebrospinal fluid

During early stages of embryo development, the brain cavity is filled with embryonic cerebrospinal fluid (E-CSF), a complex fluid containing different protein fractions that contributes to the regulation of the survival, proliferation and neurogenesis of the neuroectodermal stem cells. Using 2-DE, protein sequencing and database searches, we identified and analyzed the proteome of the E-CSF from chick embryos (Gallus gallus). We identified 26 different gene products, including proteins related to the extracellular matrix, proteins associated with the regulation of osmotic pressure and metal transport, proteins related to cell survival, MAP kinase activators, proteins involved in the transport of retinol and vitamin D, antioxidant and antimicrobial proteins, intracellular proteins and some unknown proteins. Most of these gene products are involved in the regulation of developmental processes during embryogenesis in systems other than E-CSF. Interestingly, 14 of them are also present in adult human CSF proteome, and it has been reported that they are altered in the CSF of patients suffering neurodegenerative diseases and/or neurological disorders. Understanding these molecules and the mechanisms they control during embryonic neurogenesis is a key contribution to the general understanding of CNS development, and may also contribute to greater knowledge of these human diseases.     

Neurovascular crosstalk coordinates the central nervous system development

Purpose of the review: The synchronic development of vascular and nervous systems is orchestrated by common molecules that regulate the communication between both systems. The identification of these common guiding cues and the developmental processes regulated by neurovascular communication are slowly emerging. In this review, we describe the molecules modulating the neurovascular development and their impact in processes such as angiogenesis, neurogenesis, neuronal migration, and brain homeostasis. Recent findings: Blood vessels not only are involved in nutrient and oxygen supply of the central nervous system (CNS) but also exert instrumental functions controlling developmental neurogenesis, CNS cytoarchitecture, and neuronal plasticity. Conversely, neurons modulate CNS vascularization and brain endothelial properties such as blood–brain barrier and vascular hyperemia. Summary: The integration of the active role of endothelial cells in the development and maintenance of neuronal function is important to obtain a more holistic view of the CNS complexity and also to understand how the vasculature is involved in neuropathological conditions.     

Neuronal action on the developing blood vessel pattern

The nervous system relies on a highly specialized network of blood vessels for development and neuronal survival. Recent evidence suggests that both the central and peripheral nervous systems (CNS and PNS) employ multiple mechanisms to shape the vascular tree to meet its specific metabolic demands, such as promoting nerve-artery alignment in the PNS or the development the blood brain barrier in the CNS. In this article we discuss how the nervous system directly influences blood vessel patterning resulting in neuro-vascular congruence that is maintained throughout development and in the adult.     

Impact of manganese on and transfer across blood-brain and blood-cerebrospinal fluid barrier in vitro

Manganese occupational and dietary overexposure has been shown to result in specific clinical central nervous system syndromes, which are similar to those observed in Parkinson disease. To date, modes of neurotoxic action of Mn are still to be elucidated but are thought to be strongly related to Mn accumulation in brain and oxidative stress. However, the pathway and the exact process of Mn uptake in the brain are yet not fully understood. Here, two well characterized primary porcine in vitro models of the blood-brain and the blood-cerebrospinal fluid (CSF) barrier were applied to assess the transfer of Mn in the brain while monitoring its effect on the barrier properties. Thus, for the first time effects of MnCl(2) on the integrity of these two barriers as well as Mn transfer across the respective barriers are compared in one study. The data reveal a stronger Mn sensitivity of the in vitro blood-CSF barrier compared with the blood-brain barrier. Very interestingly, the negative effects of Mn on the structural and functional properties of the highly Mn-sensitive blood-CSF barrier were partly reversible after incubation with calcium. In summary, both the observed stronger Mn sensitivity of the in vitro blood-CSF barrier and the observed site-directed, most probably active, Mn transport toward the brain facing compartment, reveal that, in contrast to the general assumption in literature, after oral Mn intake the blood-CSF barrier might be the major route for Mn into the brain.     

Novel insights into the development and maintenance of the blood–brain barrier

The blood–brain barrier (BBB) is essential for maintaining homeostasis within the central nervous system (CNS) and is a prerequisite for proper neuronal function. The BBB is localized to microvascular endothelial cells that strictly control the passage of metabolites into and out of the CNS. Complex and continuous tight junctions and lack of fenestrae combined with low pinocytotic activity make the BBB endothelium a tight barrier for water soluble moleucles. In combination with its expression of specific enzymes and transport molecules, the BBB endothelium is unique and distinguishable from all other endothelial cells in the body. During embryonic development, the CNS is vascularized by angiogenic sprouting from vascular networks originating outside of the CNS in a precise spatio-temporal manner. The particular barrier characteristics of BBB endothelial cells are induced during CNS angiogenesis by cross-talk with cellular and acellular elements within the developing CNS. In this review, we summarize the currently known cellular and molecular mechanisms mediating brain angiogenesis and introduce more recently discovered CNS-specific pathways (Wnt/β?catenin, Norrin/Frizzled4 and hedgehog) and molecules (GPR124) that are crucial in BBB differentiation and maturation. Finally, based on observations that BBB dysfunction is associated with many human diseases such as multiple sclerosis, stroke and brain tumors, we discuss recent insights into the molecular mechanisms involved in maintaining barrier characteristics in the mature BBB endothelium.     

Quantitative and noninvasive assessment of prenatal X-ray-induced CNS abnormalities using magnetic resonance imaging

Our purpose was to noninvasively assess formation of the microvasculature, blood-brain barrier (BBB) and blood-CSF barrier formation of prenatal X-ray-induced CNS abnormalities using quantitative MRI. Eight pregnant female Sprague-Dawley rats were divided into two groups consisting of control and X-irradiated animals. After birth, 20 neonatal male rats were divided into four groups of five rats. To evaluate the development of the BBB, changes in T(1) induced by Gd-DTPA were compared quantitatively in normal and prenatally irradiated animals in the formative period 1 to 2 weeks after birth. To assess the abnormalities of the microvasculature, quantitative perfusion MRI and MR angiography were also used. Histology was also performed to evaluate the BBB (albumin) and vascular endothelial cells (laminin). Decreased cerebral blood flow (CBF) and angioarchitectonic abnormalities were observed in the prenatally irradiated rats. However, abnormalities of the BBB and blood-CSF barrier were not observed using Gd-enhanced MRI and albumin staining. Quantitative perfusion MRI, MR angiography and Gd-enhanced T(1) mapping are useful for assessing CNS disturbance after prenatal exposure to radiation. These techniques provide important diagnostic information for assessing the condition of patients during the early stages of life after accidental or unavoidable prenatal exposure to radiation.     

Delayed astrocytic contact with cerebral blood vessels in FGF‐2 deficient mice does not compromise permeability properties at the developing blood‐brain barrier

The brain functions within a specialized environment tightly controlled by brain barrier mechanisms. Understanding the regulation of barrier formation is important for understanding brain development and may also lead to finding new ways to deliver pharmacotherapies to the brain; access of many potentially promising drugs is severely hindered by these barrier mechanisms. The cellular composition of the neurovascular unit of the blood‐brain barrier proper and their effects on regulation of its function are beginning to be understood. One hallmark of the neurovascular unit in the adult is the astroglial foot processes that tightly surround cerebral blood vessels. However their role in barrier formation is still unclear. In this study we examined barrier function in newborn, juvenile and adult mice lacking fibroblast growth factor‐2 (FGF‐2), which has been shown to result in altered astroglial differentiation during development. We show that during development of FGF‐2 deficient mice the astroglial contacts with cerebral blood vessels are delayed compared with wild‐type animals. However, this delay did not result in changes to the permeability properties of the blood brain barrier as assessed by exclusion of either small or larger sized molecules at this interface. In addition cerebral vessels were positive for tight‐junction proteins and we observed no difference in the ultrastructure of the tight‐junctions. The results indicate that the direct contact of astroglia processes to cerebral blood vessels is not necessary for either the formation of the tight‐junctions or for basic permeability properties and function of the blood‐brain barrier. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1201–1212, 2016     

The role of blood-brain barrier in the development of childhood febrile seizures and temporal lobe epilepsy

The blood-brain barrier (BBB) of the central nervous system (CNS) is a physiological barrier that makes it possible to control the exchange of ions, molecules and cells between blood and brain tissue and prevent their free inflow into the brain. BBB is crucial for maintenance of brain homeostasis. The BBB damage accompanies many degenerative, neurological and inflammatory (infectious or noninfectious) diseases and pathological states. Current review reports about the BBB role in the development of childhood febrile seizures and temporal lobe epilepsy.     

SSeCKS regulates angiogenesis and tight junction formation in blood-brain barrier

The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and low permeability. BBB maintenance is important in the central nervous system (CNS) because disruption of the BBB may contribute to many brain disorders, including Alzheimer disease and ischemic stroke. The molecular mechanisms of BBB development remain ill-defined, however. Here we report that src-suppressed C-kinase substrate (SSeCKS) decreases the expression of vascular endothelial growth factor (VEGF) through AP-1 reduction and stimulates expression of angiopoietin-1 (Ang-1), an antipermeability factor in astrocytes. Conditioned media from SSeCKS-overexpressing astrocytes (SSeCKS-CM) blocked angiogenesis in vivo and in vitro. Moreover, SSeCKS-CM increased tight junction proteins in endothelial cells, consequently decreasing [3H]sucrose permeability. Furthermore, immunoreactivity to SSeCKS gradually increased during the BBB maturation period, and SSeCKS-expressing astrocytes closely interacted with zonula occludens (ZO)-1-expressing blood vessels in vivo. Collectively, our results suggest that SSeCKS regulates BBB differentiation by modulating both brain angiogenesis and tight junction formation.     

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The human central nervous system (CNS) vasculature expresses a distinctive barrier phenotype, the blood–brain barrier (BBB). As the BBB contributes to low efficiency in CNS pharmacotherapy by restricting drug transport, the development of an in vitro human BBB model has been in demand. Here, we present a microfluidic model of CNS angiogenesis having three-dimensional (3D) lumenized vasculature in concert with perivascular cells. We confirmed the necessity of the angiogenic tri-culture system (brain endothelium in direct interaction with pericytes and astrocytes) to attain essential phenotypes of BBB vasculature, such as minimized vessel diameter and maximized junction expression. In addition, lower vascular permeability is achieved in the tri-culture condition compared to the monoculture condition. Notably, we focussed on reconstituting the functional efflux transporter system, including p-glycoprotein (p-gp), which is highly responsible for restrictive drug transport. By conducting the calcein-AM efflux assay on our 3D perfusable vasculature after treatment of efflux transporter inhibitors, we confirmed the higher efflux property and prominent effect of inhibitors in the tri-culture model. Taken together, we designed a 3D human BBB model with functional barrier properties based on a developmentally inspired CNS angiogenesis protocol. We expect the model to contribute to a deeper understanding of pathological CNS angiogenesis and the development of effective CNS medications.     

One messenger shared by two systems: How cytokines directly modulate neurons

Cytokines are small, secreted proteins that are known for their roles in the immune system. An accumulating body of evidence indicates that cytokines also work as neuromodulators in the central nervous system (CNS). Cytokines can access the CNS through multiple routes to directly impact neurons. The neuromodulatory effects of cytokines maintain the overall homeostasis of neural networks. In addition, cytokines regulate a diverse repertoire of behaviors both at a steady state and in inflammatory conditions by acting on discrete brain regions and neural networks. In this review, we discuss recent findings that provide insight into how combinatorial codes of cytokines might mediate neuro-immune communications to orchestrate functional responses of the brain to changes in immunological milieus.     

Retinoic acid signaling in vascular development

Formation of the vasculature is an essential developmental process, delivering oxygen and nutrients to support cellular processes needed for tissue growth and maturation. Retinoic acid (RA) and its downstream signaling pathway is vital for normal pre‐ and post‐natal development, playing key roles in the specification and formation of many organs and tissues. Here, we review the role of RA in blood and lymph vascular development, beginning with embryonic yolk sac vasculogenesis and remodeling and discussing RA's organ‐specific roles in angiogenesis and vessel maturation. In particular, we highlight the multi‐faceted role of RA signaling in CNS vascular development and acquisition of blood–brain barrier properties.     

Neurovascular Unit: a Focus on Pericytes

The blood–brain barrier (BBB) is a highly specialized system that controls the exchanges between the blood and the central nervous system (CNS). This barrier shields the CNS from toxic substances in the blood and provides nutrients to CNS, thus playing an essential role in the maintenance of homeostasis. The anatomical basis of the BBB is formed by the endothelial cells of brain microvasculature, with elaborated tight and adherens junctions, which together with pericytes, the basement membrane, and astrocytes, as well as neurons, microglia and oligodendrocytes form the neurovascular unit. The interaction between all these components guarantees a proper environment for neural function and a restricted permeability and transport. Pericytes were initially reported by Rouget in 1873 and since then they have been recognized as an important component of the BBB, despite the difficulty of their identification. Diverse functions have been assigned to pericytes, including a role in BBB properties, hemostasis, and angiogenesis, as well as a contractile, immune, and phagocytic function. These cells are also seen like multipotent cells and so with a great potential for therapy. Here, we review the neurovascular unit composition and the interplay between the diverse components, addressing pericytes with a particular detail.     

Shiga toxin-2 enhances heat-shock-induced apoptotic cell death in cultured and primary glial cells

The blood–brain barrier (BBB) selectively controls the homeostasis of the central nervous system (CNS) environment using specific structural and biochemical features of the endothelial cells, pericytes, and glial limitans. Glial cells, which represent the cellular components of the mature BBB, are the most numerous cells in the brain and are indispensable for neuronal functioning. We investigated the effects of Shiga toxin on glial cells in vitro. Shiga toxin failed to inhibit cell proliferation but attenuated expression of heat shock protein 70, which is one of the chaperone proteins, in cultured and primary glial cells. Furthermore, the combination of Shiga toxin and a heat shock procedure induced cell apoptosis and decreased cell proliferation in both cells. Thus, we speculate that glial cell death in response to the combination of Shiga toxin and heat shock might weaken the BBB and induce central nervous system complications.     

Neuronal--glial interactions during development and aging.

Integration of the central nervous system is an expression of cerebral homeostasis that is essential for the internal ability of the organism to adapt to its changing environment throughout life. It is generally accepted that neurons undergo no further division after differentiation, whereas glial cells continue to proliferate throughout life. The increase in glial cells with advanced age may reflect a compensatory process of the brain to overcome neuronal loss or neuronal functional changes that may occur with age. Therefore, these neuronal-glial interactions during development and aging may play a key role in the integrative capacity of the brain. One of the mechanisms contributing to brain stability is the blood-brain barrier, which regulates the neuronal-glial microenvironment in the mature organism. Neuronal intercommunication is mediated via neurotransmitter substances and a shift may occur from excitation to inhibition and vice versa in some CNS areas with aging. Studies of some aspects of cholinergic, monoaminergic and amino acid neurotransmission show that their maturational patterns are CNS-area specific and that some neurotransmitter processes decline with advanced age. Glial cells, besides participating in the regulation of extraneuronal environment, are also proposed to be involved in neurotransmission mechanisms in the adult and aging CNS and since they are the major CNS cellular compartment that changes with age they may thus contribute significantly to the maintenance of CNS integrative ability and adaptation with age.