Download Perivascular Spaces and the Two Steps to Neuroinflammation

J Neuropathol Exp Neurol
Copyright Ó 2008 by the American Association of Neuropathologists, Inc.
Vol. 67, No. 12
December 2008
pp. 1113Y1121
REVIEW ARTICLE
Perivascular Spaces and the Two Steps to Neuroinflammation
Trevor Owens, PhD, Ingo Bechmann, MD, and Britta Engelhardt, PhD
Abstract
Immune cells enter the central nervous system (CNS) from the
circulation under normal conditions for immunosurveillance and in
inflammatory neurologic diseases. This review describes the distinct
anatomic features of the CNS vasculature that permit it to maintain
parenchymal homeostasis and which necessitate specific mechanisms for neuroinflammation to occur. We review the historical
evolution of the concept of the blood-brain barrier and discuss
distinctions between diffusion/transport of solutes and migration of
cells from the blood to CNS parenchyma. The former is regulated at
the level of capillaries, whereas the latter takes place in postcapillary
venules. We summarize evidence that entry of immune cells into the
CNS parenchyma in inflammatory conditions involves 2 differently
regulated steps: transmigration of the vascular wall into the
perivascular space and progression across the glia limitans into the
parenchyma.
Key Words: Blood-brain barrier, Endothelial cell, Glia limitans,
Neuroinflammation, Neurovascular unit, Perivascular space.
INTRODUCTION
The interface between the immune and nervous systems has long fascinated both immunologists and neuroscientists. This is best exemplified at the most prominent
interface between these systems, the blood-brain barrier
(BBB). In discussions of clinical and experimental inflammatory brain diseases, brain homeostasis, and psychology,
the BBB takes on almost mystical significance. It is striking,
however, how many different versions of the BBB one can
encounter in such discussions, depending on the focus of the
discussants. We and others have reviewed this topic in other
forums but felt it would be useful to focus on the anatomic
and physiologic intricacies of the BBB with reference to its
pathobiologic functions. We emphasize the concept that
cellular transmigration across the BBB is not a single-step
phenomenon as occurs in other tissues, but that it involves 2
distinct processes that overlap within the perivascular space.
This 2-step nature of cellular migration into the central
nervous system (CNS) imposes specific constraints that are
fundamental to understanding many facets of neuroimmunology and CNS pathophysiology.
From the Medical Biotechnology Center, University of Southern Denmark
(TO), Odense C, Denmark; Institute for Clinical Neuroanatomy, Johann
Wolfgang Goethe-University (IB), Frankfurt, Germany; and Theodor
Kocher Institute, University of Bern (BE), Bern, Switzerland.
Send correspondence and reprint requests to: Trevor Owens, PhD, Medical
Biotechnology Center, University of Southern Denmark, Winsloewparken
25, 5000 Odense C, Denmark; E-mail: [email protected]
Morphology of the Neurovascular Unit: Vascular
Wall, Perivascular Space, and Glia Limitans
The topography and architecture of CNS microvessels
are designed for 2 major site-specific tasks. First, because
neuronal activity within the CNS strictly depends on homeostasis, the microvasculature of the brain must prevent ions and
other solutes in the blood from entering the parenchyma.
Second, to enhance regional blood flow wherever it is needed,
the vascular tree has to have the capacity to respond rapidly to
neuronal activity. The first task is reflected by the term BBB;
the second is referred to as Bneurovascular coupling.[ Both the
differentiation of BBB-typical endothelial cells and the response of the vessels to enhanced electric activity seem to
depend in various ways on astrocyte signaling provided at a
unique structure, the glia limitans (Fig. 1). The glia limitans is
composed of astrocyte processes and covers the entire surface
of the brain and spinal cord on the external face, toward the
subarachnoid space (glia limitans superficialis), and internally
around the vessels (glia limitans perivascularis; Fig. 2A).
By definition, capillaries in all organs lack a tunica
media of smooth muscle cells. Capillary endothelial cells in
the CNS are surrounded by large numbers of pericytes that are
embedded into the vascular basement membrane, which is
directly attached to the glia limitans (Fig. 2C). This contrasts
with CNS arteries and veins in which the distance between
the endothelial layer and the glia limitans is much larger because there are various layers of intervening smooth muscle
cells. In postcapillary CNS venules, there is an intermediate
situation with a perivascular space and the presence of Bmural
cells[ within the endothelial basement membrane (Fig. 2B);
these have characteristics of pericytes or vascular smooth
muscle cells (1).
As they pass from the subarachnoid space into the deep
areas of the brain, the large vessels are surrounded by a connective tissues consisting of leptomeningeal mesothelial cells,
macrophages, and other antigen-presenting cells comprising
the cellular components of the perivascular space (Fig. 2A).
Thus, perivascular spaces are located between the basement
membrane of the vascular wall and the basement membrane
of the glia limitans and are continuous with the subarachnoid
space (Fig. 1B). In the capillary segment, the basement
membranes contain laminins 411 and 511, whereas in the glia
limitans, they contain laminins 111 and 211; the latter fuse
to form a Bgliovascular membrane[ that closes the perivascular space (Fig. 2D) (2). As a consequence, a unidirectional
flow of extracellular fluids from the parenchyma along
perivascular spaces into the subarachnoid space can be driven
by the arterial pulse; this substitutes for the lack of lymphatic
drainage in the brain (3).
J Neuropathol Exp Neurol Volume 67, Number 12, December 2008
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Owens et al
J Neuropathol Exp Neurol Volume 67, Number 12, December 2008
FIGURE 1. Glia limitans and perivascular spaces. (A) Immunostaining for glial fibrillary acidic protein highlights the ‘‘glia
limitans perivascularis’’ in a human brain. (B) In standard histologic sections, perivascular spaces are not apparent around
capillaries (arrows) but are evident (asterisk) where capillaries
merge to form postcapillary venules.
Where/What is the BBB?
The term BBB describes the phenomenon that was first
observed by Paul Ehrlich (4) in the course of his studies with
Bintravital dyes.[ These hydrophilic compounds can be injected into the blood and change their color depending on
their oxidative state. Ehrlich’s goal was to compare the oxygen consumption of different organs based on the color they
exhibited upon dye injection. A prerequisite of this approach
was that the dye should be equally distributed within the
body at the beginning of the experiment, but Ehrlich noted
that this was not the case. In particular, the brain exhibited
little or no staining. He therefore concluded that the brain
lacks affinity for these dyes. Continuing with this work, Biedl
and Kraus (5) injected bile acids into the CNS, and the Berlin
physician Lewandowski (6) injected sodium ferrocyanide
into the CNS, from which he concluded that the Bcapillary
wall must block the entrance of certain molecules[ not
normally present in the blood. This was the first time that a
special barrier function was postulated for brain capillary
endothelial cells.
The development of electron microscopy allowed further focus on the Bbarrier.[ Reese and Karnovsky (7) were the
first to describe the unique belt-like tight junctions in brain
FIGURE 2. (A) Schematic diagram of different anatomic regions from brain parenchyma to skull. Superficial vessels of the brain [6]
are located in the subarachnoid space [7]. This compartment is delineated by the arachnoid mater [4] and the pia mater [3]. The
surface of the brain is completely covered by the astrocytic endfeet of the glia limitans [2]. Toward the subarachnoid space, these
endfeet are designated as glia limitans superficialis (A); toward the vessels inside of the brain, they are termed glia limitans
perivascularis (B). On their way from the surface to the deep areas of the brain, the vessels take leptomeningeal connective tissue
with them, thereby forming perivascular spaces [10], which remain connected to the subarachnoid space. 1 indicates perikaryon
of an astrocyte; 2, glia limitans superficialis; 3, connective tissue of the pia mater (inner layer of the leptomeninges); 4, arachnoid
mater (outer layer of the leptomeninges); 5, subarachnoid connective tissue (trabeculae arachnoideae); 6, subarachnoid vessel; 7,
subarachnoid space; 8, dura mater (pachymeninges); 9, neurothelium; 10, perivascular space; 11, penetrating vessel; 12, capillary; 13, glia limitans perivascularis. (B, C) Ultrastructure of the PVS, which is bordered by the other walls of a postcapillary venule
and the glia limitans (arrowheads) or their basement membranes. The field corresponds to the white bar (A). (C) Higher
magnification of the field depicted (B) shows the basement membranes. In noncapillary vessels, at least 3 basement membranes
can be distinguished: the endothelial membrane (dotted line), the outer vascular membrane (dashed line), and the membrane of
the glia limitans (dotted and dashed). (D) Ultrastructure of the region corresponding to the black bar (A). (D) In capillaries (12 in
[A]), the basement membranes are merged to form a ‘‘fused gliovascular membrane’’ that occludes the perivascular space. (E)
Higher magnification of the field depicted (D). The capillary wall consists of endothelium [E], endothelial basement membrane
(dotted line), and Pe. The fused gliovascular membrane is shown by a continuous black line. It is directly apposed to the glia
limitans. Astrocyte processes of the glia limitans are indicated by arrowheads. The arrows (C, E) point to tight junctions between
endothelial cells. The overlap of adjacent endothelial cells is a hallmark of the BBB and is evident in the capillary, but not in the
venule. E, endothelial cell; L, lumen; MC, mural cell, an intermediate form between smooth muscle cell and pericyte; Pe, pericytes;
PVS, perivascular space. Figure reprinted from Radivoj V. Krstic: Die Gewebe des Menschen und der Säugetiere (Human and
Mammalian Tissues), 1988, with kind permission of Springer Science + Business Media.
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J Neuropathol Exp Neurol Volume 67, Number 12, December 2008
capillary endothelial cells, which they regarded as correlates
of a BBB. In addition, they noted Ba paucity[ of transportation vesicles that linked the concept of a barrier to more
dynamic processes. Indeed, we now know that in addition to
the mechanical barrier maintained by tight junctions at the
capillary level, a number of permanently active transportation
mechanisms contribute to excluding blood molecules that
could harm the parenchymal milieu necessary for neural
transmission (8).
The term BBB thus provides a metaphorical explanation for a phenomenon first noted by Ehrlich rather than
pointing to a single anatomic structure or mechanism. In fact,
much confusion comes from attempts to translate the concept of a BBB into the field of cellular neuroinflammation.
Whereas the permission or restriction of passage of molecules
from the blood is a major function of capillaries, immune cells
are recruited to the CNS at the level of postcapillary venules;
the architecture and topographic relationships of these vessels
to the brain parenchyma are strikingly different due to the
existence of mural cells and perivascular spaces. The width of
perivascular spaces is variable in the healthy CNS (Fig. 1B).
Distance from the glia limitans seems to affect astrocyte signaling on endothelial cell differentiation; and therefore,
notable differences have been described in the organization
of tight junctions in capillaries compared with precapillary
and postcapillary vessels (Figs. 2C, E) (9). Unfortunately, a
systematic ultrastructural study of the molecular composition
of tight junctions in capillaries versus in postcapillary venules
has not yet been performed. It is not generally appreciated
that it is only at first glance that markers of the BBB such as
horseradish peroxidase (HRP) do not leave the blood stream
and enter the brain when they are injected into the circulation
(10). A closer look reveals that HRP staining can be observed
in the vascular wall of larger vessels and in macrophages of
the perivascular spaces and the choroid plexus, which thus
act as substitutes of BBB function at the precapillary and
postcapillary levels; their capacity to act as phagocytic scavengers compensates for the enhanced endothelial layer permeability in these areas of the vascular tree. This additional
level of regulation of diffusion from the blood into the
parenchyma of the brain prompted Goldmann (11) to propose
the notion of a Bphysiologic bordering membrane[ consisting
of highly phagocytic macrophages in 1913. Since then, the
penetration of BBB markers through noncapillary brain
vessels has remained the neuroanatomists’ Bdirty little secret[
(12, 13). Nevertheless, it can be concluded that the mechanisms that maintain a barrier for solutes in postcapillary
venules involve additional components.
From studies of bone marrow chimeras, it became
evident that the population of perivascular macrophages is
regularly exchanged by hematogenous precursors in the absence of pathologic processes in the brain parenchyma
(14Y16). In light of the more recent appreciation of the
effects of irradiation on cellular recruitment into the brain
(17), it is noteworthy that this exchange also occurs in
nonirradiated rats (18). Thus, monocytes can apparently cross
the vascular wall of brain vessels under normal conditions.
They do not, however, normally progress through the glia
limitans into the neuropil, but are retained in perivascular
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Perivascular Spaces in Neuroinflammation
spaces. Only when there is intraparenchymal damage such as
axonal degeneration or inflammation, as in multiple sclerosis
(MS) or its animal model experimental autoimmune encephalomyelitis (EAE), does the glia limitans become permissive for monocytes to migrate into the neuropil; some of them
may then transform into microglia (15, 19, 20). For example,
Babcock et al (21) showed that the chemokine monocyte
chemotactic protein 1 (CC-ligand 2) produced by astrocytes
and microglia is a critical factor driving the recruitment of
monocytes into areas of Wallerian degeneration.
Two distinct steps can also be distinguished for T-cell
migration into the CNS parenchyma. One group also demonstrated that the elimination of perivascular macrophages
did not interfere with perivascular infiltration after the
induction of EAE in mice, but the progression of T cells
across the glia limitans was completely blocked (22). Thus,
macrophages and/or antigen recognition seem to be required
for T cells to perform the second step of migration. The
Angelov group showed that sensitizing rats with HRP and
subsequently injecting HRP into their subarachnoid space led
to diffusion along perivascular spaces and phagocytosis of
HRP by perivascular macrophages. When the animals were
immunologically boosted with HRP, T cells entered the
perivascular spaces but did not cross the glia limitans; they
were essentially put Bon hold[ in the perivascular compartment (23). Similarly, deletion of both matrix metalloprotease
(MMP) 2 and MMP-9 confers resistance to EAE by trapping
leukocytic infiltrates in the perivascular space, suggesting
that MMPs produced by macrophages are required for
leukocyte infiltration across the glia limitans (24).
Thus, the term BBB in the context of infiltration of
leukocytes into the CNS parenchyma can be confusing.
Physiologically, the term refers to the vascular segment of the
capillaries that regulate diffusion of solutes, whereas in an
inflammatory response, the term refers to the postcapillary
venules, that is, the vessels from which leukocytes migrate
into the CNS. These are distinct vascular segments (25). For
leukocytes to reach the CNS parenchyma, they need to
perform 2 differently regulated steps: first, to cross the vascular wall, and second, to traverse the glia limitans (Fig. 3).
Understanding the distinct molecular mechanisms is critical
because it seems that activated lymphocytes regularly
penetrate the endothelial barrier for immunosurveillance of
the CNS, but only upon penetration of the glia limitans and
infiltration of the CNS parenchyma do leukocytes come into
direct contact with the parenchyma, which leads to clinical
disease.
Migration Across the Vascular Wall and
(Sometimes) Progression Across the
Glia Limitans
As previously described, there are 2 principal physical
barriers to cellular entry to the CNS parenchyma, the vascular
endothelium and the glia limitans, each with its own
basement membrane. The perivascular space between them
represents not so much a barrier as a regulatory checkpoint
that depends on the precise anatomic localization and the
immune status of the patient or animal.
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Owens et al
J Neuropathol Exp Neurol Volume 67, Number 12, December 2008
FIGURE 3. The 2 steps to neuroinflammation. (A) In the first
step of neuroinflammation (1st), circulating immune cells cross
the microvascular central nervous system (CNS) endothelium
to reach the PVS. In the second step (2nd), immune cells
progress across the glia limitans to enter the CNS Pa. (B, C)
The distinction between perivascular and intraparenchymal
infiltration in standard histologic sections in the spinal cord of
a mouse with EAE. (B) In a paraffin section, the border built by
the glia limitans has been traversed by inflammatory cells on
the right side (2nd), whereas cells are restricted to the
perivascular space on the left side (1st). (C) Phase-contrast
microscopy highlights the distinction. The arrowheads point
to 2 cells that appear to ‘‘digest’’ or otherwise ‘‘open’’ the
basement membrane of the glia limitans (white line), which is
not visible at the light microscopic level. Pa, parenchyma; PVS,
perivascular space.
Migration of leukocytes across the vascular wall is
governed by adhesion molecules, cytokines, chemokines, and
their receptors. These are integrated in a sequential process of
leukocyte-endothelial cell interaction that was first formulated by Butcher (26) and Dustin and Springer (27). The
molecular mechanisms involved in the multistep paradigm of
leukocyte recruitment across the vascular wall have been
extensively reviewed (26Y29). Briefly, leukocytes in the socalled Bfast lane[ of in the vessel lumens engage with
inflamed endothelium via tethering and rolling (i.e. slowing)
and are triggered by chemokine gradients to higher affinity
adhesion, locomotion, and frank adhesion before they cross
the vascular wall (diapedesis; Fig. 4).
CNS Address and Welcome Mats
Tissue-specific Barea codes[ are combinations of
adhesion and signaling ligands that are expressed selectively
or at higher levels in the CNS vasculature and are thought to
favor interactions with leukocytes that express the specific
selection of appropriate counterligands. The area code
concept would explain selective migration of leukocytes to
different anatomic regions such as the CNS, although the
precise molecular composition of CNS area codes has not yet
been defined. At present, there are no CNS-specific endothelial cell adhesion ligands that are absolutely specific. Certain
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combinations, however, can favor leukocyte interactions
with cerebral or spinal cord endothelial cells. For example,
whereas most circulating leukocytes express the adhesion
molecule L-selectin ligand (CD62-L), a subset of activated or
memory-effector T cells have downregulated L-selectin.
Therefore, they are less likely to reenter lymph nodes from
the circulation, and their migration may favor the CNS. The
selectins and the selectin ligand P-selectin glycoprotein
ligand 1 are not required for leukocyte infiltration across
CNS microvessels such as in EAE (29, 30), and in mice, a
counterligand for >4-integrins, vascular cell adhesion molecule 1, is constitutively expressed on spinal cord endothelial
cells and mediates the capture and firm adhesion of T-cell
blasts via >4-integrin (31, 32).
Selectins are a family of carbohydrate-recognizing cell
surface proteins (calcium-dependent C-type lectins) that are
widely expressed both by leukocytes and endothelial cells.
Selectin-driven tethering promotes rolling and permits the
slowed-down leukocytes to be triggered by local gradients of
immobilized chemokines. The process is selective in that
those blood cells with appropriate chemokine receptors will
then proceed to subsequent integrin activation (inside-out
signaling), locomotion to sites of entry, and tight adhesion
(28). The fact that selectins are not equally required for
tethering adhesion in the inflamed CNS vessels influences the
design of therapies directed against entry of potentially
pathogenic leukocytes (29, 33).
Integrin interactions mediate firm leukocyte adhesion
and are therefore likely targets for therapy. The efficacy of
>4-integrinYdirected immunotherapy in MS represents an
important proof-of-principle in this regard because it opens
up possibilities that other adhesion moleculeYligand interactions may be useful drug targets (31). The recent
identification of a role for the adhesion ligandYactivated
leukocyte cell adhesion molecule or CD166 in CD6+ CD4+
T-cell interactions with cerebral endothelial cells is a
potential candidate for this therapeutic approach (34).
Integration of Autoimmune and Infectious
Immune Principles
The multistep paradigm is also applicable to immune
responses to CNS infections. In the skin, local virus infections induce the trafficking of antigen-presenting Langerhans cells to lymph nodes where, as dendritic cells, they
induce a memory-effector T-cell response that is then
directed to the site of infection via local inflammation (28).
A role for local inflammation in this process is noteworthy. In
contrast, EAE in rodents can be induced by adoptive transfer
to an unmanipulated recipient of activated CD4+ T cells that
have specificity for myelin peptides. Alternatively, it can be
induced by subcutaneous immunization with these myelin
peptides in adjuvant in susceptible rodent strains. Although
many of the actively induced EAE models involve pertussis
toxin, it is not needed for disease induction either by
immunization or in adoptive transfer in certain rodent strains.
It may, however, increase the disease incidence (35); thus, in
these models, a CNS-specific disease can be induced without
the use of adjuvant or preexisting local inflammation. One
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Perivascular Spaces in Neuroinflammation
FIGURE 4. Multistep paradigm for leukocyte entry to the central nervous system (CNS). In the first step of neuroinflammation
circulating, immune cells engage in a multistep interaction with CNS microvascular endothelial cells that they cross by either
transcellular or paracellular routes. After passing through the endothelial basement membrane (EBM), they reach the perivascular
space (PVS). After additional activation triggers that are probably provided by perivascular macrophages or dendritic cells, MMP-2
and MMP-9 are produced and allow the second-step immune cell entry across the glia limitans or pavenchymal basement
membrane (PBM) into the CNS parenchyma.
interpretation of these observations is that whether leukocyte
interaction with CNS endothelium results in extravasation or
disease may be determined by the sum of the molecular
interactions between them. This presumes that highly
activated T cells that have sufficiently elevated levels of
adhesion molecules and cytokine/chemokine production can
elicit responses from an otherwise quiescent vascular endothelium. In support of this concept, T-cell diapedesis across
noninflamed retinal microvessels fails until the interaction
has induced endothelial intercellular cell adhesion molecule 1
expression, which probably triggers the extravasation (36).
Moreover, T-cellYderived tumor necrosis factor can elicit a
chemokine response from BBB-associated cells, principally
dendritic cells/macrophages/pericytes/microglial cells (37).
The activation status of the T cell is critical to this process
because only freshly activated T cells can transfer EAE and
productively interact with the noninflamed BBB (32). The
extent to which activated T cells and local inflammation (e.g.
resulting from infection or other local CNS responses) may
be involved in CNS inflammation in MS is not known.
In Vitro Models
In vitro models of CNS endothelium have resulted in
many advances in our understanding of the BBB. Specific in
vitro models mimic many aspects of live animals, but a full
neurovascular unit is too complex to reproduce in vitro at
present. Cocultures of cerebral endothelial cells with apposed
astrocytes that induce a tight junctional barrier in vitro have
been designed to model the capillary BBB and investigate the
passage of solutes across this vascular bed (37). Recently, these
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systems have been adapted to model postcapillary venules
where astrocytes and endothelia are not in intimate contact,
that is, more closely mimicking postcapillary venules (32).
Exchange and Turnover of Cells in the
Perivascular Space
Cellular migration across the BBB is recognized as a
critical step for CNS inflammation. Seminal studies by
Hickey and Kimura (14) showed that for induction of EAE,
encephalitogenic T cells and bone marrow-derived cells in
CNS, presumed to be at the BBB, should share expression
of major histocompatibility complex (MHC), or be MHCcompatible. Further studies by Hickey et al (38) and by
Sedgwick et al (39) determined that a population of perivascular macrophages is replaced in radiation chimeras,
distinct from parenchymal microglia, which are not. This
observation points to the ability of blood-derived cells to exchange within the perivascular space, that is, at least some
blood-derived cells cross the vascular endothelium under
normal circumstances. These would be the relevant MHCmatched cells with which T cells must interact. This is
consistent with the observation that dendritic cells, which are
usually found in extraparenchymal locations (e.g. the perivascular and subarachnoid spaces and meninges), are critical
for the induction of EAE (40).
Is There Antigen-Driven Interaction Between
T Cells and Endothelia?
It is uncertain whether endothelial cells present antigen
to CD4+ T cells for the induction of CNS inflammation. The
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Owens et al
relative absence of expression of the CD4 ligand MHC II on
normal endothelium and the fact that EAE can be adoptively
transferred to naive recipients suggest that antigen-driven
interactions between CD4+ T cells and endothelial cells are
not necessary for their extravasation. The logic behind this
argument contributes to interpretation of data (41, 42) showing that naive CD4+ T cells can access the CNS. Endothelial
cells can be induced to express MHC II and costimulatory
molecules in vitro, and this may identify the capacity of
endothelial cells to present antigen. Some observations in
situ, however, do not support this (43, 44), suggesting more
cautious interpretation. Expression of such molecules was
increased in disease-associated endothelial inflammation in
autopsy material (45), but this may represent a post hoc
effect relative to the initial T-cell entry that is the focus here.
The observation that adhesion of CD4+ T cells is increased
when activated endothelial cells express costimulatory molecules CD40, B7.1/CD80, or B7.2/CD86 (46, 47) likely has
functional significance but does not prove antigen recognition in vivo.
A constitutive low level of the CD8 ligand MHC I on
cerebral endothelium suggests that migrating CD8+ T cells
may engage in antigen recognition (48). In that study, naive
CD8+ T cells were detected in the side of the brain in which
cognate peptide had been injected, and this was blocked by
antibody against MHC I, which could only access the luminal
endothelial surface (48). The authors’ interpretation of the
absence of T cells in the contralateral brain, in which an
irrelevant antigen was injected, that CD8+ T cells could only
enter if antigen was presented, is valid only if a transient
Blook-and-leave[ entry can be excluded. The issue turns on
whether egress or reverse migration contributes to net effects
(see following).
Emigration
Egress or reverse migration is difficult to measure (49).
One recent study described cells that were assumed to have
left the CNS (50), but consideration of cellular exit from the
CNS can confound interpretation of in vivo analyses. For
example, the failure to detect T cells within the CNS cannot
be taken to show that they did not enter the CNS. When T
cells are detected, it is presumed to be the net result of entry
plus either proliferation or accumulation (51). The concept of
immunosurveillance predicts that potentially protective T
cells should be able to access all tissues, which can be extended to include that in the absence of functional antigen
presentation, they may either leave again or die in situ (veni,
vidi, vici or veni, mori [52]).
Transcellular Versus Paracellular Routes
of Migration
Leukocytes may go through endothelial cells (transcellular route) or go between them (paracellular route). Transcellular migration was originally described by Marchesi and
Gowans (53) in electron microscope studies that showed
Bemperipolesis,[ whereby lymphocytes were enveloped by
cells at vascular walls. Which molecular mechanisms trigger
transcellular versus paracellular diapedesis is presently
unclear; the use of both routes in different vascular beds
has been described for both lymphocytes and neutrophils
(53, 54). In the CNS, the passage between endothelial cells
would necessitate the disruption of the complex tight junctions; molecular interactions with the junctional molecules
claudins, occludin, junctional adhesion molecules, CD99,
and Zonula occludens molecules would be involved (54).
Although there is accumulating evidence for transcellular
TABLE. Summary of Functional and Structural Characteristics of the Capillary and Post-capillary Blood-Brain Barrier
Capillaries
Post-capillary Venules
Definition
Physiological BBB: Diffusion barrier for solutes
Diameter
Continuous endothelium
Endothelium-specific transporters, carriers, channels,
cytoplasmic enzymes
Pinocytotic
Leukocyte traffic signals (adhesion molecules,
chemokines)
Endothelial cell tight junctions
Endothelial basement membrane
Pericytes (embedded in endothelial basement
membrane)
Smooth muscle cells (beyond endothelial basement
membrane)
Perivascular space
Perivascular macrophages or DC
Glia limitans; parenchymal basement membrane and
astrocytic endfeet
e6 Km
Present
All present
Neuroimmunological BBB: diapedesis of immune
cells
e50 Km
Present
Partially present (i.e. transferrin receptor [55]),
but mostly unknown
Low, but higher than in capillary endothelium
Present (32)
Low
Partially present
Highly complex
Present
PDGFR-A-positive, NG2-positive,
desmin-positive pericytes present (56)
Absent
Absent
Absent
Present
Complex
Present
PDGFR-A-positive, NG2-postive, desmin-positive
pericytes present
>-SMC actin-positive cells present? (Absent at the
ultrastructural level)
Present
Present
Present
*, Abbreviations: NG2, nerve/glial antigen-2; PDGFR-A, platelet-derived growth factor receptor-A SMC, smooth muscle cell.
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J Neuropathol Exp Neurol Volume 67, Number 12, December 2008
diapedesis of leukocytes across the BBB during inflammation
(54), the molecular mechanisms remain to be investigated.
The functional and structural characteristics.
Migration Across the Glia Limitans and
Its Regulation
The glia limitans seems to be the site at which MMPs
play the most significant role in leukocyte migration. ToftHansen et al (57) showed that, whereas a broad-spectrum
MMP inhibitor did not affect the subclinical perivascular
accumulation of leukocytes in a transgenic mouse expressing
the chemokine CC-ligand 2 in the CNS, MMP inhibition
blocked leukocyte entry into CNS parenchyma induced by
pertussis toxin. This indicates that chemokine-driven crossing
of the endothelium and perivascular leukocyte accumulation
was not dependent on MMPs, whereas they were critical for
migration across the glia limitans. The specific MMPs
involved were not identified in that study. As previously
mentioned, however, in EAE, MMP-2 and MMP-9 proteolytically cleave dystroglycan, which anchors astrocyte endfeet to the glia limitans basement membrane via binding to
the extracellular matrix molecules agrin and perlecan (24).
Moreover, staining for the transmembrane A-dystroglycan
isoform is lost in the pertussis toxinYtreated myelin basic
protein promoterYdriven CC-ligand 2 transgenic mice, indicating that dystroglycan breakdown occurs in this model
(Füchtbauer et al, unpublished observation).
The glia limitans is not as accessible to in vitro analysis
as the cerebrovascular endothelium. Nevertheless, there
currently is a consensus that chemokines and integrins exert
their major effects at the vascular endothelium, whereas
MMPs have more prominent roles in glia limitans breakdown. Astrocytes are a potential source of chemokines at
the BBB and probably contribute significantly to perivascular chemokine levels under physiologic conditions (58, 59).
Another contribution that astrocytes make is modifying
overall BBB permeability. Several groups have shown that
permeability of artificial BBB endothelial monolayers to
macromolecules is significantly decreased by coculture with
astrocytes or by treatment with astrocyte-derived soluble
factors (60, 61). In a recent study that showed expression of
angiotensin receptors at the BBB in MS brain and on
endothelial cells in vitro, Wosik et al (62) reported that
astrocytes can modify expression and phosphorylation of the
endothelial cell tight junctionYassociated molecule occludin
via release of angiotensin. Rodent astrocytes have also been
reported to express AT1 angiotensin receptors and may respond to vascular tension-regulatory hormones (63Y65).
The BloodYCerebrospinal Fluid Barrier as
Possible Entry Site of Immune Cells into the CNS
In contrast to the classical endothelial BBB, the
epithelial bloodYcerebrospinal fluid (CSF) barrier of the
choroid plexus epithelium has not, until very recently, been
considered to be an entry site for immune cells or pathogens
into the CNS (66). The choroid plexus extends from the
ventricular surface into the lumen of the ventricles; its major
known function is the secretion of CSF. It is organized in a
villous surface, including an extensive microvascular netÓ 2008 American Association of Neuropathologists, Inc.
Perivascular Spaces in Neuroinflammation
work enclosed by a single layer of cuboidal epithelium (67).
The microvessels within the choroid plexus parenchyma
differ from those of the brain parenchyma because they allow
free movement of molecules via fenestrations and intercellular gaps (68). The functional barrier between the blood
compartment and the CSF is located at the level of the
choroid plexus epithelial cells that form tight junctions that
inhibit paracellular diffusion of water-soluble molecules (69).
These tight junctions are the morphologic correlate of the
blood-CSF barrier and have a unique molecular composition
of transmembrane proteins with occludin, claudin-1 and
claudin-2, and claudin-11 (69).
Regional Differences in CNS Microvessels:
Parenchyma, Leptomeninges, and
Circumventricular Organs
In addition to the choroid plexus, there are structures in
the CNS of mammals that lack an endothelial BBB. These
areas are commonly referred to as the circumventricular
organs (CVOs) and perform homeostatic and neurosecretory
functions; the neurons within them monitor hormonal stimuli
and other substances within the blood or secrete neuroendocrines into the blood (70, 71). Circumventricular organs
are localized at strategic points close to the midline of the
brain within the ependymal walls lining the third and fourth
ventricles. Because they lack an endothelial BBB, they lie
within the blood milieu and thus form a blood-CSF barrier.
Similar to the choroid plexus, a complex network of tight
junctions connecting specialized ependymal cells (tanycytes)
seal off the CNS from the CVOs (71, 72). Although the
CVOs have often been referred to as Bwindows of the brain[
with respect to soluble mediators, entry of immune cells into
the brain via the CVOs across the tanycytic barriers has not
been investigated extensively to date (73).
In addition to the obvious differences in fenestrated
capillaries within the CVOs and the barrier-forming microvessels within the CNS, regional differences in the characteristics of the BBB have also been described. For example,
mouse endothelioma cells established from the cerebellum
respond in a more pronounced fashion than cerebral endotheliomas to proinflammatory cytokines by increased permeability and reduced transendothelial electrical resistance (74).
Molecular and cellular differences between meningeal
and parenchymal microvascular beds have also been
described (75, 76); meningeal microvessels lack astrocytic
ensheathment. Interestingly, the differences between meningeal and parenchymal CNS blood vessel endothelial cells
extend to differences in their expression patterns of P- and
E-selectin. In the healthy CNS, meningeal blood vessel endothelial cells can be distinguished from those in the CNS
parenchyma by their constitutive expression of P-selectin,
which is absent from endothelial cells of parenchymal blood
vessels (30, 77, 78). Furthermore, injection of proinflammatory cytokines into mice induced expression of E-selectin in
meningeal but not in parenchymal CNS blood vessel endothelial cells (79). Thus, leukocytes that extravasate across
meningeal vessels arrive in the leptomeningeal space from
which they might travel within the perivascular spaces; if
confronted with an inflammatory stimulus, they could enter
1119
Copyright @ 2008 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 67, Number 12, December 2008
Owens et al
the CNS parenchyma. Meningeal blood vessels have been
described as being leakier than parenchymal blood vessels
and more reactive to proinflammatory cytokines, and this
might contribute to leukocyte recruitment restricted to the
meningeal compartment.
CONCLUSION
In summary, we have proposed that the term BBB is
somewhat of a misnomer. The barrier concept is more
applicable to solute entry, the neuroinflammatory relevance
of which relates more to edema than to cellular migration.
The process of leukocyte entry into the CNS parenchyma is
controlled by different cellular components at the level of
postcapillary venules. Thus, we highlight the concept that
regulation of cellular entry involves migration across 2
distinct structures, the vascular wall and the glia limitans
and their associated basement membranes. This migration
involves 2 steps for the development of neuroinflammation.
ACKNOWLEDGMENTS
The authors acknowledge discussions within the COST
Action BM0603 Inflammation in Brain Disease Neurinfnet
and networking support from COST.
REFERENCES
1. Gerhardt H, Betsholtz C. Endothelial-pericyte interactions in angiogenesis. Cell Tissue Res 2003;314:15Y23
2. Sixt M, Engelhardt B, Pausch F, Hallmann R, Wendler O, Sorokin LM.
Endothelial cell laminin isoforms, laminins 8 and 10, play decisive roles
in T cell recruitment across the blood-brain barrier in experimental
autoimmune encephalomyelitis. J Cell Biol 2001;153:933Y46
3. Carare RO, Bernardes-Silva M, Newman TA, et al. Solutes, but not
cells, drain from the brain parenchyma along basement membranes of
capillaries and arteries: Significance for cerebral amyloid angiopathy and
neuroimmunology. Neuropathol Appl Neurobiol 2008;34:131Y44
4. Ehrlich P. Das Sauerstoff-Bedürfnis des Organismus. Eine farbenanalytische Studie. [On the oxygen consumption of the body. A study using
intravital dyes.]. Berlin: Verlag von August Hirschwald, 1885
5. Biedl A, Kraus R. Über eine bisher unbekannte toxische Wrikung der
Gallensäuren auf das Zentralnervensystem. Zentralblatt Innere Medizin
1898;19:1185Y200
6. Lewandowski M. Zur Lehre von der Cerebrospinalflüssigkeit. [On the
cerebrospinal fluid]. Zentralblatt Klein Forschung 1900;40:480Y94
7. Reese TS, Karnovsky MJ. Fine structural localization of a blood-brain
barrier to exogenous peroxidase. J Cell Biol 1967;34:207Y17
8. Zlokovic BV. The blood-brain barrier in health and chronic neurodegenerative disorders. Neuron 2008;57:178Y201
9. Ge S, Song L, Pachter JS. Where is the blood-brain barrier...really?
J Neurosci Res 2005;79:421Y27
10. Kristensson K, Olsson Y. Accumulation of protein tracers in pericytes of
the central nervous system following systemic injection in immature
mice. Acta Neurol Scand 1973;49:189Y94
11. Goldmann E. Vitalfärbungen am Zentralnervensystem. Beitrag zur
Physio-Pathologie des Plexus Choroideus und der Hirnhäute. [Intravital
labelling of the central nervous system. A study on the pathophysiology
of the choroid plexus and the meninges.] Abhandlungen der königlich
preubischen Akademie der Wissenschaften Physikalisch-Mathematische
Classe 1913;1:1Y64
12. Galea I, Bechmann I, Perry VH. What is immune privilege (not)? Trends
Immunol 2007;28:12Y18
13. Janeway CA Jr. Approaching the asymptote evolution and revolution in
immunology. Cold Spring Harb Symp Quant Biol 1989;54:1Y13
14. Hickey WF, Kimura H. Perivascular microglial cells of the CNS are
bone marrowYderived and present antigen in vivo. Science 1988;239:
290Y92
1120
15. Priller J, Flugel A, Wehner T, et al. Targeting gene-modified hematopoietic cells to the central nervous system: Use of green fluorescent
protein uncovers microglial engraftment. Nat Med 2001;7:1356Y61
16. Bechmann I, Priller J, Kovac A, et al. Immune surveillance of mouse
brain perivascular spaces by blood-borne macrophages. Eur J Neurosci
2001;14:1651Y58
17. Mildner A, Schmidt H, Nitsche M, et al. Microglia in the adult brain
arise from Ly-6ChiCCR2+ monocytes only under defined host conditions. Nat Neurosci 2007;10:1544Y53
18. Bechmann I, Kwidzinski E, Kovac AD, et al. Turnover of rat brain
perivascular cells. Exp Neurol 2001;168:242Y49
19. Bechmann I, Goldmann J, Kovac AD, et al. Circulating monocytic cells
infiltrate layers of anterograde axonal degeneration where they transform
into microglia. FASEB J 2005;19:647Y49
20. Ladeby R, Wirenfeldt M, Garcia-Ovejero D, et al. Microglial cell
population dynamics in the injured adult central nervous system. Brain
Res Brain Res Rev 2005;48:196Y206
21. Babcock AA, Kuziel WA, Rivest S, Owens T. Chemokine expression by
glial cells directs leukocytes to sites of axonal injury in the CNS.
J Neurosci 2003;23:7922Y30
22. Tran EH, Hoekstra K, van Rooijen N, Dijkstra CD, Owens T. Immune
invasion of the central nervous system parenchyma and experimental
allergic encephalomyelitis, but not leukocyte extravasation from blood,
are prevented in macrophage-depleted mice. J Immunol 1998;161:
3767Y75
23. Walther M, Popratiloff A, Lachnit N, et al. Exogenous antigen containing perivascular phagocytes induce a non-encephalitogenic extravasation of primed lymphocytes. J Neuroimmunol 2001;117:30Y42.
24. Agrawal S, Anderson P, Durbeej M, et al. Dystroglycan is selectively
cleaved at the parenchymal basement membrane at sites of leukocyte
extravasation in experimental autoimmune encephalomyelitis. J Exp
Med 2006;203:1007Y19
25. Bechmann I, Galea I, Perry VH. What is the blood-brain barrier (not)?
Trends Immunol 2007;28:5Y11
26. Butcher EC. Leukocyte-endothelial cell recognition: Three (or more)
steps to specificity and diversity. Cell 1991;67:1033Y36
27. Dustin ML, Springer TA. Role of lymphocyte adhesion receptors in
transient interactions and cell locomotion. Annu Rev Immunol 1991;9:
27Y66
28. von Andrian UH, Mackay CR. T-Cell function and migration. Two sides
of the same coin. N Engl J Med 2000;343:1020Y34
29. Engelhardt B, Ransohoff RM. The ins and outs of T-lymphocyte
trafficking to the CNS: Anatomical sites and molecular mechanisms.
Trends Immunol 2005;26:485Y95
30. Doring A, Wild M, Vestweber D, Deutsch U, Engelhardt B. E- and
P-selectin are not required for the development of experimental
autoimmune encephalomyelitis in C57BL/6 and SJL mice. J Immunol
2007;179:8470Y79
31. Man S, Ubogu EE, Ransohoff RM. Inflammatory cell migration into the
central nervous system: A few new twists on an old tale. Brain Pathol
2007;17:243Y50
32. Vajkoczy P, Laschinger M, Engelhardt B. Alpha4-integrinYVCAM-1
binding mediates G proteinYindependent capture of encephalitogenic T
cell blasts to CNS white matter microvessels. J Clin Invest 2001;108:
557Y65
33. Engelhardt B, Kempe B, Merfeld-Clauss S, et al. P-selectin glycoprotein
ligand 1 is not required for the development of experimental autoimmune encephalomyelitis in SJL and C57BL/6 mice. J Immunol 2005;
175:1267Y75
34. Cayrol R, Wosik K, Berard JL, et al. Activated leukocyte cell adhesion
molecule promotes leukocyte trafficking into the central nervous system.
Nat Immunol 2008;9:137Y45
35. Munoz JJ, Bernard CC, Mackay IR. Elicitation of experimental allergic
encephalomyelitis (EAE) in mice with the aid of pertussigen. Cell
Immunol 1984;83:92Y100
36. Xu H, Manivannan A, Liversidge J, Sharp PF, Forrester JV, Crane IJ.
Requirements for passage of T lymphocytes across non-inflamed retinal
microvessels. J Neuroimmunol 2003;142:47Y57
37. Murphy CA, Hoek RM, Wiekowski MT, Lira SA, Sedgwick JD.
Interactions between hemopoietically derived TNF and central nervous
Ó 2008 American Association of Neuropathologists, Inc.
Copyright @ 2008 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 67, Number 12, December 2008
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
system-resident glial chemokines underlie initiation of autoimmune
inflammation in the brain. J Immunol 2002;169:7054Y62
Hickey WF, Vass K, Lassmann H. Bone marrow-derived elements in
the central nervous system: An immunohistochemical and ultrastructural survey of rat chimeras. J Neuropathol Exp Neurol 1992;
51:246Y56
Sedgwick JD, Schwender S, Imrich H, Dorries R, Butcher GW, ter
Meulen V. Isolation and direct characterization of resident microglial
cells from the normal and inflamed central nervous system. Proc Natl
Acad Sci U S A 1991;88:7438Y42
Greter M, Heppner FL, Lemos MP, et al. Dendritic cells permit immune
invasion of the CNS in an animal model of multiple sclerosis. Nat Med
2005;11:328Y34
Krakowski ML, Owens T. Naive T lymphocytes traffic to inflamed
central nervous system, but require antigen recognition for activation.
Eur J Immunol 2000;30:1002Y9
Bauer J, Bradl M, Hickey WF, et al. T-Cell apoptosis in inflammatory
brain lesions: Destruction of T cells does not depend on antigen
recognition. Am J Pathol 1998;153:715Y24
Pedersen EB, McNulty JA, Castro AJ, Fox LM, Zimmer J, Finsen B.
Enriched immune-environment of blood-brain barrier deficient areas of
normal adult rats. J Neuroimmunol 1997;76:117Y31
Redwine JM, Buchmeier MJ, Evans CF. In vivo expression of major
histocompatibility complex molecules on oligodendrocytes and neurons
during viral infection. Am J Pathol 2001;159:1219Y24
van der Maesen K, Hinojoza JR, Sobel RA. Endothelial cell class II
major histocompatibility complex molecule expression in stereotactic
brain biopsies of patients with acute inflammatory/demyelinating
conditions. J Neuropathol Exp Neurol 1999;58:346Y58
Omari KM, Chui R, Dorovini-Zis K. Induction of beta-chemokine
secretion by human brain microvessel endothelial cells via CD40/CD40L
interactions. J Neuroimmunol 2004;146:203Y8
Quandt J, Dorovini-Zis K. The beta chemokines CCL4 and CCL5
enhance adhesion of specific CD4 + T cell subsets to human brain
endothelial cells. J Neuropathol Exp Neurol 2004;63:350Y62
Galea I, Bernardes-Silva M, Forse PA, van Rooijen N, Liblau RS, Perry
VH. An antigen-specific pathway for CD8 T cells across the blood-brain
barrier. J Exp Med 2007;204:2023Y30
Cserr HF, Knopf PM. Cervical lymphatics, the blood-brain barrier and
the immunoreactivity of the brain: A new view. Immunol Today 1992;
13:507Y12
Goldmann J, Kwidzinski E, Brandt C, Mahlo J, Richter D, Bechmann I.
T cells traffic from brain to cervical lymph nodes via the cribroid plate
and the nasal mucosa. J Leukoc Biol 2006;80:797Y801
Owens T, Renno T, Taupin V, Krakowski M. Inflammatory cytokines in
the brain: Does the CNS shape immune responses? Immunol Today
1994;15:566Y71
Lehmann PV. The fate of T cells in the brain: Veni, vidi, vici and veni,
mori. Am J Pathol 1998;153:677Y80
Marchesi VT, Gowans JL. The migration of lymphocytes through the
endothelium of venules in lymph nodes: An electron microscope study.
Proc R Soc Lond B Biol Sci 1964;159:283Y90
Engelhardt B, Wolburg H. Mini-review: Transendothelial migration of
leukocytes: Through the front door or around the side of the house? Eur
J Immunol 2004;34:2955Y63
Kissel K, Hamm S, Schulz M, Vecchi A, Garlanda C, Engelhardt B.
Immunohistochemical localization of the murine transferrin receptor
(TfR) on blood-tissue barriers using a novel anti-TfR monoclonal
antibody. Histochem Cell Biol 1998;110:63Y72
Dore-Duffy P. Pericytes: Pluripotent cells of the blood brain barrier. Curr
Pharm Des 2008;14:1581Y93
Toft-Hansen H, Buist R, Sun XJ, Schellenberg A, Peeling J, Owens T.
Metalloproteinases control brain inflammation induced by pertussis toxin
Ó 2008 American Association of Neuropathologists, Inc.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
Perivascular Spaces in Neuroinflammation
in mice overexpressing the chemokine CCL2 in the central nervous
system. J Immunol 2006;177:7242Y49
Farina C, Aloisi F, Meinl E. Astrocytes are active players in cerebral
innate immunity. Trends Immunol 2007;28:138Y45
Ransohoff RM, Kivisakk P, Kidd G. Three or more routes for leukocyte
migration into the central nervous system. Nat Rev Immunol 2003;3:
569Y81
Janzer RC, Raff MC. Astrocytes induce blood-brain barrier properties in
endothelial cells. Nature 1987;325:253Y57
Prat A, Biernacki K, Wosik K, Antel JP. Glial cell influence on the
human blood-brain barrier. Glia 2001;36:145Y55
Wosik K, Cayrol R, Dodelet-Devillers A, et al. Angiotensin II controls
occludin function and is required for blood brain barrier maintenance:
Relevance to multiple sclerosis. J Neurosci 2007;27:9032Y42
Sumners C, Tang W, Zelezna B, Raizada MK. Angiotensin II receptor
subtypes are coupled with distinct signal-transduction mechanisms in
neurons and astrocytes from rat brain. Proc Natl Acad Sci U S A 1991;
88:7567Y71
Li J, Culman J, Hortnagl H, et al. Angiotensin AT2 receptor protects
against cerebral ischemia-induced neuronal injury. FASEB J 2005;19:
617Y19
Gebke E, Muller AR, Jurzak M, Gerstberger R. Angiotensin IIYinduced
calcium signalling in neurons and astrocytes of rat circumventricular
organs. Neuroscience 1998;85:509Y20
Engelhardt B, Wolburg-Buchholz K, Wolburg H. Involvement of the
choroid plexus in central nervous system inflammation. Microsc Res
Tech 2001;52:112Y29
Dziegielewska KM, Ek J, Habgood MD, Saunders NR. Development of
the choroid plexus. Microsc Res Tech 2001;52:5Y20
Betz LA, Goldstein GW, Katzman R. Blood-brainYcerebrospinal fluid
barriers. In: Siegel GJ, ed. Basic Neurochemistry: Molecular, Cellular,
and Medical Aspects. 4th Ed. New York: Raven Press, 1989:591Y606
Wolburg H, Wolburg-Buchholz K, Liebner S, Engelhardt B. Claudin-1,
claudin-2 and claudin-11 are present in tight junctions of choroid plexus
epithelium of the mouse. Neurosci Lett 2001;307:77Y80
Johnson AK, Gross PM. Sensory circumventricular organs and brain
homeostatic pathways. FASEB J 1993;7:678Y86
Leonhardt H. Ependym und circumventriculäre Organe. In: Oksche A,
Vollrath L, eds. Handbuch der mikroskopischen Anatomie des Menschen. Berlin Heidelberg New York: Springer, 1980:177Y666
Bouchaud C, Bosler O. The circumventricular organs of the mammalian
brain with special reference to monoaminergic innervation. Int Rev
Cytol 1986;105:283Y327
Schulz M, Engelhardt B. The circumventricular organs participate in the
immunopathogenesis of experimental autoimmune encephalomyelitis.
Cerebrospinal Fluid Res 2005;2:8
Silwedel C, Forster C. Differential susceptibility of cerebral and
cerebellar murine brain microvascular endothelial cells to loss of barrier
properties in response to inflammatory stimuli. J Neuroimmunol 2006;
179:37Y45
Allt G, Lawrenson JG. Is the pial microvessel a good model for
blood-brain barrier studies? Brain Res Brain Res Rev 1997;24:67Y76
Rascher G, Wolburg H. The tight junctions of the leptomeningeal
blood-cerebrospinal fluid barrier during development. J Hirnforsch 1997;
38:525Y40
Carrithers MD, Visintin I, Kang SJ, Janeway CA Jr. Differential
adhesion molecule requirements for immune surveillance and inflammatory recruitment. Brain 2000;123:1092Y101
Barkalow FJ, Goodman MJ, Gerritsen ME, Mayadas TN. Brain
endothelium lack one of two pathways of P-selectinYmediated neutrophil
adhesion. Blood 1996;88:4585Y93
Gotsch U, Jager U, Dominis M, Vestweber D. Expression of P-selectin
on endothelial cells is upregulated by LPS and TNF-alpha in vivo. Cell
Adhes Commun 1994;2:7Y14
1121
Copyright @ 2008 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.