Blood-Brain Barrier

Previously we have employed AntiBodies to the Tight Junction (TJ)-associated proteins ZO-1 and Occludin to describe Endothelial Tight Junction abnormalities, in lesional and Normal-Appearing White Matter, in Primary and Secondary/Progressive Multiple Sclerosis (MS).

This work is extended here by use of AntiBodies to the independent TJ-specific proteins and Junctional Adhesion Molecule A & B (JAM-A, JAM-B). We have also assessed the expression in MS of beta-Catenin, a protein specific to the TJ-associated Adherens Junction.

ImmunoCytoChemistry and semiquantitative confocal microscopy for JAM-A and beta-Catenin was performed on snap-frozen sections from MS cases (n = 11) and controls (n = 6).

Data on 1,443 blood vessels was acquired from active Lesions (n = 13), inactive lesions (n = 13), NAWM (n = 20) and control White Matter (n = 13).

In MS abnormal JAM-A expression was found in active (46%) and inactive lesions (21%), comparable to previous data using ZO-1.

However, a lower level of TJ abnormality was found in MS NAWM using JAM-A (3%) compared to ZO-1 (13%).

JAM-B was strongly expressed on a small number of large blood vessels in control and MS tissues but at too low a level for quantitative analysis.

By comparison with the high levels of abnormality observed with the TJ proteins, the Adherens Junction protein beta-Catenin was normally expressed in all MS and control tissue categories.

These results confirm, by use of the independent marker JAM-A, that TJ abnormalities are most frequent in active White Matter lesions.

Altered expression of JAM-A, in addition to affecting junctional tightness may also both reflect and affect Leukocyte trafficking, with implications for Immune status within the diseased CNS.

Conversely, the Adherens Junctions component of the TJ, as indicated by beta-Catenin expression is normally expressed in all MS and control tissue categories.

The Blood-Brain Barrier (BBB) is a complex organization of Cerebral Endothelial Cells (CEC), Pericytes and their basal lamina, which are surrounded and supported by Astrocytes and PeriVascular Macrophages.

Collectively these cells separate and form the compartments of the Cerebral Vascular space and the Cerebral Interstitium under normal conditions.

Without the BBB, the 'interior milieu' of the Central Nervous System (CNS) would be flooded by Humoral NeuroTransmitters and formed blood elements that upset normal CNS functions and lead to Vascular/Neural Injury.

Dysregulation of the BBB and TransEndothelial migration of activated Leukocytes are among the earliest CerebroVascular abnormalities seen in Multiple Sclerosis (MS) Brains and parallel the release of inflammatory Cytokines/Chemokines.

Mechanisms for breakdown of the BBB in MS are incompletely understood, but appear to involve direct effects of these Cytokines/ Chemokines on Endothelial regulation of BBB components, as well as indirect Cytokine/Chemokine-dependent Leukocyte mediated injury.

Unique Endothelial structural features of the BBB include highly organized Endothelial Tight Junctions, the absence of Class II Major Histocompatibility Complex, abundant Mitochondria and a highly developed transport system in CEC.

Exposure of Endothelium to ProInflammatory Cytokines (IFN-γ, TNF- and IL-1ß) interrupts the BBB by disorganizing cell-cell junctions, decreases the Brain solute barrier, enhances Leukocyte Endothelial Adhesion and migration.

As well as increases expression of Class II MHC and promotes shedding of Endothelial 'MicroParticles' (EMP).

In this review we examine interactions between Cytokines/Chemokines, activated Leukocytes, Adhesion Molecules and activated CEC in the pathogenesis of BBB failure in MS.

Blood-Brain Barrier (BBB) HyperPermeability in Multiple Sclerosis (MS) is associated with lesion pathogenesis and has been linked to pathology in MicroVascular Tight Junctions (TJs).

This study quantifies the uneven distribution of TJ pathology and its association with BBB leakage.

Frozen sections from plaque and Normal-Appearing White Matter (NAWM) in 14 cases were studied together with White Matter from six neurological and five normal controls.

Using single and double immunofluorescence and confocal microscopy, the TJ-associated protein Zonula Occludens-1 (ZO-1) was examined across lesion types and tissue categories, and in relation to Fibrinogen leakage.

Confocal image data sets were analysed for 2198 MS and 1062 control vessels. Significant differences in the incidence of TJ abnormalities were detected between the different lesion types in MS and between MS and control White Matter.

These were frequent in oil-red O (ORO)(+) active plaques, affecting 42% of vessel segments, but less frequent in ORO(-) inactive plaques (23%), NAWM (13%), and normal (3.7%) and Neurological controls (8%).

A similar pattern was found irrespective of the vessel size, supporting a causal role for diffusible inflammatory mediators.

In both NAWM and inactive lesions, dual labelling showed that vessels with the most TJ abnormality also showed most Fibrinogen leakage.

This was even more pronounced in active lesions, where 41% of vessels in the highest grade for TJ alteration showed severe leakage.

It is concluded that disruption of TJs in MS, affecting both paracellular and transcellular paths, contributes to BBB leakage.

TJ abnormality and BBB leakage in inactive lesions suggests either failure of TJ repair or a continuing pathological process.

In NAWM, it suggests either pre-lesional change or secondary damage. Clinically inapparent TJ pathology has prognostic implications and should be considered when planning disease-modifying therapy.

Copyright 2003 John Wiley & Sons, Ltd.

Brain Capillary Endothelial Cells form the Blood-Brain Barrier. They are connected by extensive Tight Junctions, and are polarized into Luminal (Blood-facing) and AbLuminal (Brain-facing) plasma membrane domains.

The polar distribution of transport proteins allows for active regulation of Brain ExtraCellular Fluid.

Experiments on isolated membrane vesicles from Capillary Endothelial Cells of bovine Brain demonstrated the polar arrangement of Amino Acid and Glucose transporters, and the utility of such arrangements have been proposed.

For instance, passive carriers for Glutamine and Glutamate have been found only in the Luminal membrane of Blood-Brain Barrier Cells, while Na-dependent secondary active transporters are at the AbLuminal membrane.

This organization could promote the net removal of Nitrogen-rich Amino Acids from Brain, and account for the low level of Glutamate penetration into the Central Nervous System.

Furthermore, the presence of a gamma-Glutamyl cycle at the Luminal membrane and Na-dependent Amino Acid transporters at the AbLuminal membrane may serve to modulate movement of Amino Acids from Blood-to-Brain. Passive carriers facilitate Amino Acid transport into Brain.

However, activation of the gamma-Glutamyl cycle by increased plasma Amino Acids is expected to generate OxoProline within the Blood-Brain Barrier.

OxoProline stimulates secondary active Amino Acid transporters (Systems A and B(o)+) at the AbLuminal membrane, thereby reducing net influx of Amino Acids to Brain.

Finally, passive Glucose transporters are present in both the Luminal and AbLuminal membranes of the Blood-Brain Barrier.

Interestingly, a high affinity Na-dependent Glucose carrier has been described only in the AbLuminal membrane. This raises the question whether Glucose entry may be regulated to some extent.

Immunoblotting studies suggest more than one type of passive Glucose transporter exist in the Blood-Brain Barrier, each with an asymmetrical distribution.

In conclusion, it is now clear that the Blood-Brain Barrier participates in the active regulation of Brain ExtraCellular Fluid, and that the diverse functions of each plasma membrane domain contributes to these regulatory functions.

The Blood-Brain Barrier (BBB) is formed by Brain Endothelial Cells lining the Cerebral MicroVasculature, and is an important mechanism for protecting the Brain from fluctuations in plasma composition.

And, from circulating agents such as NeuroTransmitters and XenoBiotics capable of disturbing Neural function.

The Barrier also plays an important role in the homeostatic regulation of the Brain MicroEnvironment necessary for the stable and co-ordinated activity of Neurons.

The BBB phenotype develops under the influence of associated Brain Cells, especially Astrocytic Glia, and consists of more complex Tight Junctions than in other Capillary Endothelia

And, a number of specific Transport and Enzyme Systems which regulate molecular traffic across the Endothelial Cells.

Transporters characteristic of the BBB phenotype include both uptake mechanisms (e.g. GLUT-1 Glucose carrier, L1 Amino Acid transporter) and efflux transporters (e.g. P-GlycoProtein).

In addition to a role in long-term Barrier induction and maintenance, Astrocytes and other cells can release chemical factors that modulate Endothelial permeability over a time-scale of seconds to minutes.

Cell culture models, both primary and cell lines, have been used to investigate aspects of Barrier induction and modulation.

Conditioned medium taken from growing Glial Cells can reproduce some of the inductive effects, evidence for involvement of diffusible factors.

However, for some features of Endothelial differentiation and induction, the ExtraCellular Matrix plays an important role.

Several candidate molecules have been identified, capable of mimicking aspects of Glial-Mediated Barrier induction of Brain Endothelium; these include TGF-beta, GDNF, bFGF, IL-6 and steroids.

In addition, factors secreted by Brain Endothelial Cells including Leukaemia Inhibitory Factor (LIF) have been shown to induce Astrocytic differentiation. Thus Endothelium and Astrocytes are involved in two-way induction.

Short-term modulation of Brain Endothelial permeability has been shown for a number of small chemical mediators produced by Astrocytes and other nearby cell types.

It is clear that Endothelial Cells are involved in both long- and short-term chemical communication with neighbouring cells, with the PerivVascular End Feet of Astrocytes being of particular importance.

The role of Barrier induction and modulation in normal physiology and in pathology is discussed.

Blood-Brain Barrier (BBB) breakdown, demonstrable in vivo by enhanced MRI is characteristic of new and expanding inflammatory lesions in Relapsing/Remitting and Chronic/Progressive Multiple Sclerosis (MS).

Subtle leakage may also occur in Primary/Progressive MS. However, the anatomical route(s) of BBB leakage have not been demonstrated.

We investigated the possible involvement of InterEndothelial Tight Junctions (TJ) by examining the expression of TJ proteins (Claudins and ZO-1 ) in blood vessels in active MS lesions from 8 cases of MS and in Normal-Appearing White Matter (NAWM) from 6 cases.

Blood vessels (10-50 per frozen section) were scanned using confocal laser scanning microscopy to acquire datasets for analysis.

TJ abnormalities manifested as beading, interruption, absence or diffuse CytoPlasmic localization of fluorescence, or separation of Junctions (putative opening) were frequent (affecting 40% of vessels).

In oil-red-O-positive active plaques but less frequent in NAWM (15%), and in normal (< 2%) and Neurological controls (6%).

Putatively "open" junctions were seen in vessels in active lesions and in microscopically inflamed vessels in NAWM. Dual fluorescence revealed abnormal TJs in vessels with pre-mortem Serum protein leakage.

Abnormal or open TJs, associated with inflammation may contribute to BBB leakage in enhancing MRI lesions and may also be involved in subtle leakage in non-enhancing focal and diffuse lesions in NAWM.

BBB disruption due to Tight Junctional pathology should be regarded as a significant form of tissue injury in MS, alongside DeMyelination and AxonoPathy.

The Blood-Brain Barrier (BBB) is a specialized structure of the Central Nervous System (CNS) that restricts Immune Cell migration and soluble molecule diffusion from the systemic compartment into the CNS.

Astrocytes and Microglia are resident cells of the CNS that contribute to the formation of the BBB.

In this article, we consider the influence of these Glial Cells on the Immune regulatory functions of the MicroVascular Endothelium, with special emphasis on the human BBB.

A series of in vitro studies demonstrate that soluble factors produced by Glial Cells, under basal culture conditions, help restrict development of inflammation within the CNS.

These soluble factor effects include upregulating expression of molecules including HT7, UEA-1 Lectin-binding sites, and Angiotensin Receptors that help define the phenotype of Endothelial Cells.

These factors also induce Tight Junction formation between Brain Endothelial Cells, contributing to the restricted permeability of the BBB.

In contrast, these factors have little effect on expression of molecules by ECs that either promote Lymphocyte migration, such as Chemokines and Adhesion Molecules or molecules that are required for competent Antigen presentation, such as MHC and co-stimulatory molecules.

Glial Cells that become activated in response to signals derived from the Immune System or generated within the CNS, produce an array of inflammatory molecules that increase permeability and promote Lymphocyte trafficking and persistence.

These observations emphasize the bidirectional nature of Neural-Immune interactions; this dynamic system should be amenable to therapeutic interventions.

Copyright 2001 Wiley-Liss, Inc.

Dysfunction of the Blood-Brain Barrier (BBB) is a major hallmark of MS (Multiple Sclerosis).

Studies in our laboratories over the last decade have shown that increased BBB permeability is associated with decreased expression of Tight Junction (TJ) proteins in Brain Capillary Endothelial Cells.

Results have revealed that TJ abnormalities were most common in active lesions (42% of vessels affected), but were also present in inactive lesions (23%) and in MS Normal-Appearing White Matter (13%).

Importantly, TJ abnormality was also positively associated with leakage of the Serum protein Fibrinogen which has recently been shown to be an activator of Microglia.

TJ abnormality and the resultant Vascular permeability in both lesional and non-lesional White Matter may impair tissue homoeostasis, which may have effects on disease progression, repair mechanisms and drug delivery.

The Blood-Brain Barrier (BBB) is composed mainly of specialized Endothelial Cells characterized by the presence of InterCellular Tight Junctions.

Additionally, PeriVascular Cells, Astrocytes, and surrounding basement membranes determine BBB integrity.

BBB disruption is an early phenomenon in the formation of new White Matter Multiple Sclerosis (MS) lesions; however, knowledge of the extent of BBB changes in Gray Matter MS lesions is lacking.

Here, we studied several markers for BBB integrity in well-characterized Brain Tissue of patients with MS.

Plasma protein leakage was enhanced in White Matter lesions compared with that in Normal-Appearing White Matter, whereas plasma protein leakage was absent in Gray Matter lesions.

White Matter lesions showed irregular basement membranes and Parenchyma depositions of Collagen type IV, whereas purely Gray Matter lesions lacked basement membrane alterations.

Similarly, we observed no evidence for AstroGliosis and Tight Junction changes in Cortical MS lesions.

Although BBB dysfunction is a common feature of White Matter MS lesions, Cortical MS lesions lack markers for BBB disruption or AstroGliosis.

Our data may indicate that BBB breakdown is not a critical event in the formation of Gray Matter MS lesions.

Before entering the Central Nervous System (CNS) Immune Cells have to penetrate any one of its barriers.

Namely either the Endothelial Blood-Brain Barrier, the Epithelial Blood-CerebroSpinal Fluid Barrier or the Tanycytic Barrier around the CircumVentricular Organs, all of which maintain homeostasis within the CNS.

The presence of these barriers in combination with the lack of Lymphatic vessels and the absence of classical MHC-positive Antigen Presenting Cells characterizes the CNS as an immunologically privileged site.

In Multiple Sclerosis a large number of inflammatory cells gains access to the CNS Parenchyma.

Studies performed in Experimental Autoimmune Encephalomyelitis (EAE), a rodent model for Multiple Sclerosis, have enabled us to understand some of the molecular mechanisms involved in Immune Cell entry into the CNS.

In particular, the realization that /alpha4-Integrins play a predominant role in Leukocyte trafficking to the CNS has led to the development of a novel drug for the treatment of Relapsing/Remitting Multiple Sclerosis, which targets /alpha4-Integrin mediated Immune Cell migration to the CNS.

At the same time, the involvement of other adhesion and signalling molecules in this process remains to be investigated and novel molecules contributing to Immune Cell entry into the CNS are still being identified.

The entire process of Immune Cell trafficking into the CNS is strictly controlled by the Brain Barriers not only under physiological conditions but also during NeuroInflammation, when some barrier properties are lost.

Thus, Immune Cell entry into the CNS critically depends on the unique characteristics of the Brain Barriers maintaining CNS homeostasis.