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Introduction
The central nervothat maintain homeffects of internaentrance of
plasma molecules and toxins able to induce
r constitutes thee alterations byis review.
in turn, establish the connection with the cytoskeleton.
TJproteins form strains along the intercellular junctions,creating
the connection with those of the opposing
dade de Lisboa, Avenida Professor Gama Pinto, 1649-003
Lisbon,
Portugal; Phone: 351217946449; FAX: 351217946491;
E-mail:[email protected]
Archives of Medical Researcactivation of glial cells and neural
tissue damage. Simulta-neously, they also assure the uptake of
nutrients, togetherproviding a stable environment for neural
function. CNSbarriers exist at three key sites: the epithelial
cells of thechoroid plexus, the arachnoid epithelium that lies
underthe dura mater and completely encases the brain, andthe
cerebral endothelium that constitutes the barrier
The endothelial cells of brain capillaries are considered
theanatomic basis of the BBB. The brain microvascular endo-thelial
cells (BMEC) are characterized by the presence ofelaborate
junctional complexes that are crucial players inthe maintenance of
a functional BBB. These structuresare formed by tight junctions
(TJ) in the luminal area ofthe cell membrane and by adherens
junctions (AJ) locatedin a basolateral position (4) (Figure 1). The
TJ and AJ sharecommon characteristics because they are both formed
bytransmembrane proteins linked to cytosolic proteins which,Address
reprint requests to: Maria Alexandra Brito, PhD, Research
Institute for Medicines (iMed.ULisboa), Faculdade de Farmacia,
Universi-0188-4409/$ - see frohttp://dx.doi.org/10components of the
basement membrane, which together constitute the neurovascular
unit.BBB disruption has been reported in a wide range of CNS
pathologies, with an emergingrole in the onset and disease
progression. Accordingly, recent studies revealed
vasculardysfunction in neonatal jaundice, a common pathology in the
early neonatal periodaffecting 1/10 children presenting values of
total bilirubin O17 mg/dL (291 mM). Herewe summarize the clinical
aspects of moderate to severe neonatal jaundice and providea
comprehensive review of the literature regarding bilirubin-induced
neurotoxicity froma vascular-centered approach. The collected
evidence place endothelial dysfunction andpericyte demise as key
players in the disruption of CNS homeostasis, mainly in cases
oflasting hyperbilirubinemia, thus pointing to novel targets to
prevent neurological dysfunc-tion due to severe neonatal jaundice.
2014 IMSS. Published by Elsevier Inc.Key Words: Bloodebrain
barrier, Endothelial cells, Kernicterus, Neonatal jaundice,
Neurovascularunit, Pericytes.
us system (CNS) contains cellular barrierseostasis by protecting
the brain from thel and external changes, preventing the
between blood and brain (1e3). The lattebloodebrain barrier
(BBB), of which thneonatal jaundice are the main focus of th
BBB Overviewactions with glial cells, neurons, and perivascular
pericytes as well as with the acellularelaborate junctional
complexes that nearly obliterate the intercellular space as well
asthe presence of influx and efflux transporters. Endothelial cells
establish important inter-BloodeBrain Barrier and Bilirubin: C
Maria Alexandra Brito,a,b Ine^s Palmela,a Filipa LoaResearch
Institute for Medicines (iMed.ULisboa), bDepartment of Bioc
Lisbon
Received for publication November 10, 2014; ac
The bloodebrain barrier (BBB) is a complin central nervous
system (CNS) homeostcules into the brain parenchyma and prevwhile
promoting the efflux of several mocells are the anatomical basis of
the BBBnt matter. Copyright 2014 IMSS. Published by
Elsevier.1016/j.arcmed.2014.11.015TICLE
ical Aspects and Experimental Data
co Cardoso,a Ine^s Sa-Pereira,a and Dora Britesa,b
try and Human Biology, Faculty of Pharmacy, Universidade de
Lisboa,
tugal
d November 18, 2014 (ARCMED-D-14-00652).
nd dynamic structure that plays a key roleIt strictly regulates
the entrance of mole-the access of neurotoxins and pathogensles.
The brain microvascular endothelialich has unique characteristics
such as the
h 45 (2014) 660e676Inc.
-
terpla
t jun
661BloodeBrain Barrier in Neonatal JaundiceFigure 1. Simplified
representation of the bloodebrain barrier and of the inbarrier
function of brain endothelial cells is achieved by the presence of
tighmembrane and obliterating the intercellular space (5),
anorganization that is more evident in the brain than in
otherregions of the organism (6). Examples of TJ proteins are
thecytosolic zonula occludens-1 (ZO-1) and the transmem-brane
occludin and claudin-5, as well as the recentlydiscovered
tricellulin (5). Among AJ proteins, the moststudied are the
vascular endothelial-cadherin, an adhesivetransmembrane protein,
and the cytosolic b-catenin (4,7).Alterations in TJ proteins are
directly associated with bar-rier compromise. However, AJ also play
an important rolein maintaining barrier function. In fact, they are
essentialfor TJ formation as they directly activate signaling
mole-cules and can regulate gene transcription (8e10).
Accord-ingly, changes in the expression or distribution of
AJproteins should also be considered to be involved in defectsof
the endothelial barrier (11).
BMEC are additionally characterized by a number of re-ceptors
and the presence of influx and efflux transportersthat assure the
passage of substances into and out of thebrain parenchyma. Influx
transporters are grouped accord-ingly with the type of the
transported molecule, which in-cludes energy sources, amino acids,
organic anions andneurotransmitters, among others (12). One of the
most
ability. TJ are constituted by transmembrane proteins, such as
claudins and occ
occludens (ZO) family. The adherens junctions are formed by the
transmembrane
The cytoplasmic proteins from these two types of junctions
establish the connect
brain endothelial cells to the basement membrane is mediated by
integrins in the a
of endothelial cells responsible for transcytosis. Endothelial
cells establish impo
microglia, neurons and, indirectly, with oligodendrocytes,
giving rise to the concy between the different components of the
neurovascular unit. The physical
ctions (TJ) and adherens junctions (AJ) that restrict the
paracellular perme-crucial transporters, glucose transporter-1
(GLUT-1), isresponsible for the energy supply to the brain (4).
BMECare also equipped with efflux transporters that export
un-wanted compounds from the brain parenchyma (4). Themost studied
group of efflux transporters is the ATP-binding cassette (ABC)
family that mediates the export ofsubstrates from cells coupled
with the hydrolysis of ATP(12,13), among which is the widely
studied P-glycoprotein(P-gp) (14e16).
Transport across the endothelium may additionally occurvia
vesicular mechanisms, also known as transcytosis (17).This type of
transport describes the movement of moleculeswithin endocytic
vesicles across endothelial cells, from theluminal to the abluminal
side. BMEC contain two kinds ofvesicles, clathrin-coated vesicles
and caveolae, the latterbeing the principal vesicular structure for
transcytosis inthese cells (18,19), which are schematically
depicted inFigure 1. These vesicles, also involved in various
aspectsof signal transduction (20), are plasma membrane
invagina-tions rich in caveolins, sphingolipids and
cholesterol.Caveolin-1, the major structural protein of caveolae,
is ex-pressed in several tissues with the highest levels of
expres-sion in endothelial cells, adipocytes, fibroblasts and
ludin, as well as by cytoplasmic accessory proteins including
the zonula
vascular endothelial cadherin (VE-cadherin) and the cytoplasmic
catenins.
ion to the cytoskeleton by interacting with actin filaments. The
adhesion of
bluminal surface of endothelial cells. Caveolae are membrane
invaginations
rtant communication with the basement membrane, pericytes,
astrocytes,
ept of neurovascular unit.
-
smooth-muscle cells (20). Soluble plasma molecules can be remain
to be explored. Therefore, it is fundamental to un-
662 Brito et al./ Archives of Medical Research 45 (2014)
660e676b (TGF-b) (38,39). Importantly, addition of pericytes
tocultured endothelial cells has shown to promote junctionformation
and to improve endothelial barrier function(40e44). In accordance,
several in vitro and in vivo studiesreport loss of pericytes as a
key step in brain vascular dam-age, with changes in the structure
of capillaries, in thelevels and cellular distribution of certain
junctional pro-teins, and in morphological signs of increased
endothelialpermeability (45e47). On the other hand, a link
betweenpericyte coverage and susceptibility of distinct brain
re-gions has been observed in the developing brain (48),
sug-gesting that pericyte deficiency contributes to the fragilityof
specific brain areas during development. Despite theclear role of
pericytes in BBB modulation, the signalingmechanisms involved in
the interaction of these cells withthe other components of the
barrier interface are currentlyrandomly taken up by caveolae in a
process known as bulk-phase or fluid-phase transcytosis (18,21),
which is indepen-dent of any interaction between the transported
moleculesand the vesicle membrane. In addition to ensuring
themovement of substances via transcytosis, caveolae alsohave an
active role in the regulation of BBB vesicularpermeability (20,22).
Caveolin-1 has been associated withincreased BBB permeability and
brain edema during injury(23). The effects induced by caveolin-1
seem to involve theinteraction with proteins such as vascular
endothelialgrowth factor (VEGF) receptor 2 (VEGFR-2), P-gp
andendothelial nitric oxide synthase (eNOS) (24e26). More-over,
caveolin-1 affects the expression of junctional pro-teins because
their decreased levels downregulate theexpression of occludin and
ZO-1 with important implica-tions in the regulation of permeability
(27,28).
Interactions Within the Neurovascular Unit
Endothelial cells are not alone in the formation of a properBBB.
Indeed, BMEC are surrounded by a basement mem-brane and pericytes,
which nearly embrace the capillaries intheir totality and are
further enwrapped by astrocyte feetprocesses (4,7). The interaction
of the endothelium withcellular and acellular components is crucial
for the mainte-nance of barrier properties, giving rise to the
concept ofneurovascular unit (NVU) (Figure 1).
Pericytes are cells rich in a-smooth muscle actin thatphysically
interact with the endothelium (7,29) and playan important role in
the control of blood flow (30,31). Inthe CNS, these cells present
one of the highest pericyte toBMEC ratio (1:5), when compared to
the lung (1:10) andthe skeletal muscle (1:100) (32e34), suggesting
a criticalrole in the maintenance of BBB properties. The
interactionof pericytes with BMEC is believed to control
endothelialproliferation (35), as well as the differentiation and
matura-tion of vessels (36,37), effects that are partially
regulatedthrough pericyte secretion of transforming growth
factor-derstand the multifaceted influence of pericytes not
onlyunder physiological conditions but also in the multiple
dis-orders that affect the CNS.
Another important component of the BBB is the base-ment
membrane, which surrounds both the endothelial cellsand the
pericytes (7). Because these cells are separated by asingle
basement membrane, it allows direct signalingthrough cellular
junctions (49). The basement membraneof the vasculature is a
complex assembly of four majorglycoprotein families: laminins,
collagen type IV, proteo-glycans, and nidogens (50). Extracellular
matrix (ECM)molecules are involved in cell adhesion and signaling
path-ways (51) and exist in different isoforms that can
assumeseveral combinations and differ between tissues (52).
Inaddition, some ECM molecules are associated with immunecell
recruitment to the brain parenchyma during injury(53,54). CNS
endothelial cells present several ECM recep-tors including
dystroglycan and integrins, which act to an-chor these cells to the
basement membrane. These receptorsare able to regulate BBB
integrity through the formation ofa transmembrane link between the
ECM and the endothelialcytoskeleton and by transducing
extracellular stimuli intointracellular signals (55e57). In
addition, increased integ-rin expression in endothelial cells has
been associated withcerebral blood vessel maturation during brain
development(58). Interestingly, both endothelial cells and
pericytescontribute to the synthesis of the vascular basement
mem-brane (59e61). Several in vitro studies have highlightedthe
importance of basement membrane constituents suchas fibronectin,
laminin and collagen IV in the maintenanceof the barrier integrity
and the microvessel structure(52,62). Matrix metalloproteinases
(MMPs) that can besecreted by brain cells, including endothelial
cells, maydigest the basement membrane which, in turn, leads
toreduced anchoring of the endothelium and impaired junc-tional
integrity (63,64). The complex role of the basementmembrane in
barrier integrity and cell recruitment rein-forces the importance
of considering the effects of toxicstimuli on such structure. In
addition, continuous studieson this BBB component may allow the
identification ofimportant targets for modulation in
neuropathology.
The influence of glial cells in the maintenance of aproper
barrier is fundamental, particularly when it comesto astrocytes.
The specialized foot-processes of perivascularastrocytes have a
particular role in inducing and regulatingthe BBB (65). The
positive role of astrocytes on BBB prop-erties is believed to be
achieved through the contact of theirend-feet with BMEC and through
secretion of mediators.Indeed, the presence of astrocytes or of
astrocyte-conditioned media in endothelial cultures has been
shownto promote barrier integrity and the correct assembly ofthe
intercellular junctions (42,66,67). Furthermore, astro-cytes are
able to induce the expression and polarized local-ization of
transporters including GLUT-1 (68) and
-
specialized enzyme systems (69,70). In addition, astrocytes
barrier function. These cells are responsible for the forma-
663BloodeBrain Barrier in Neonatal Jaundiceare suggested to be
necessary for the correct association ofendothelial cells and
pericytes in tube-like structures (71),indicating that interaction
among the three cell types isimportant for proper cerebral
capillary differentiation. As-trocytes are, thus, crucial players
in the formation andmaintenance of this barrier interface and are
major contrib-utors to brain homeostasis.
CNS endogenous microglia share many properties withmacrophages,
including expression of innate immune recep-tors and the ability to
phagocyte pathogens, cells or cellulardebris (72). Microglial cells
have been considered to takepart in the many interactions of BMEC
because they arefound in the perivascular space (73). However, and
despitethis proximity, their role in the regulation of BBB
propertiesis still controversial. Indeed, in vitro studies by Prat
and col-laborators (74) showed that endothelial
permeabilitydecreased in the presence of conditioned medium
collectedfrom astrocytes and microglia, probably through the
induc-tion of junction formation. Studies by Willis (75) alsoshowed
that the activation of glial cells, including micro-glia, guides
junctional proteins to paracellular domainsand restores barrier
integrity. On the contrary, activated mi-croglia were shown to
damage the endothelium as well as toinduce vascular permeability
(76e78) and transporter pro-tein dysfunction (79). Thus, microglial
cells seem to befundamental players in the modulation of BBB
properties.However, more studies on this interaction are needed
tofully clarify their influence on the NVU dynamics.
The proximity of neurons to microvessels, |15 mm (80),points to
the influence of neurons on BBB properties, butmore precise
information is needed. The neuronal influenceon BBB properties may
involve a direct interaction with theendothelium or derive from an
indirect action mediated byother brain cells such as astrocytes.
One of the striking ef-fects of neuronal activity and metabolism on
the vascula-ture is the regulation of cerebral blood flow by
amechanism known as neurovascular coupling (81,82).Some of the
acknowledged effects on the BBB attributedto neurons include the
ability to transform neuronal signalsinto vascular responses (83),
together with the induction ofbarrier integrity (84) and occludin
synthesis (85). In fact,in vitro studies with endothelial cells in
the presence of neu-rons and astrocytes highlight the importance of
both braincells in the induction of endothelial occludin
expressionand its correct localization at the membrane level
(86).The proximity of neurons to microvessels allows the
endo-thelial cells to actively participate in the regulation of
anoptimal environment for neuronal activity. In pathologieswhere
the BBB is affected, especially the barrier properties,brain
homeostasis and neuronal function often becomealtered, making the
study of this cellular interaction anessential topic to be further
explored.
Other glial cells, the oligodendrocytes, have beenignored
regarding the interactions that they may exert ontion of myelin
sheets in the CNS that are necessary for theproper conduction of
the neuronal stimuli. Interestingly,BBB influence on
oligodendrocytes has been reported,especially in demyelinating
disorders like multiple sclerosis(MS) where BBB dysfunction is
often perceived as animportant initiating step for oligodendrocyte
damage(87e89). In contrast, oligodendrocyte effect on BBB
prop-erties is still an unexplored issue. A recent study has
shownthat these glial cells are able to secrete MMP-9, which
playsan important role in the events that lead to BBB
disruptionafter white matter injury (90). In addition, in vitro
evalua-tion of the effect of oligodendrocyte-conditioned
mediumshowed that the tightness of the endothelial barrier
wasnoticeably weakened. These important findings indicatethat the
impact of oligodendrocytes on BBB properties isfar more complex
than previously thought, and this interac-tion clearly needs to be
further explored. As so, it appearsrelevant to consider
oligodendrocytes as another constituentof the NVU as we recently
proposed (7).
BBB in Neuropathology
Because BMEC are the first cells of the CNS to contactwith toxic
molecules in the systemic circulation, it is plau-sible that they
may be the first players in the signalingmechanisms that trigger
neuropathology. During injury,BMEC may produce elevated levels of
inflammatory mole-cules such as nitric oxide (NO) and cytokines
(91e95),which can influence both brain and blood compartmentsand
contribute to injury aggravation. In addition, cytokinesand other
inflammatory mediators are known to influencecaveolae, deregulating
the passage of molecules betweencompartments. They may also induce
gaps between endo-thelial cells by disassembly of intercellular
junctions, eitherthrough alterations in the cellular cytoskeleton
structure orby means of indirect damage on cell monolayer
(96,97).Accordingly, BBB disruption has been reported in a
widerange of pathologies that affect the CNS, such as MS(98,99),
Alzheimers disease (100,101), Parkinsons disease(102,103),
amyotrophic lateral sclerosis (104,105) andbrain tumors (106,107),
implicating that at some point ofthe disease progression the
release of inflammatory media-tors influence the balance between a
beneficial role in main-taining or recovering brain homeostasis and
the initiation ofneurological injury.
Neonatal Jaundice
Several diseases are particularly relevant in neonatal
lifebecause this is a period of great vulnerability for the
devel-oping CNS. Among them is neonatal hyperbilirubinemia,commonly
known as neonatal jaundice, which is consideredto be a significant
and risky condition for 1/10 children dueto elevated concentrations
of unconjugated bilirubin (UCB)(108). Although jaundice is usually
benign and normalizes
-
at the end of the first week of life, levels of total serum
bili-
0
extending beyond the first week of life and deserving acareful
management in order to prevent BIND and kernic-terus. In the same
Figure 2 (black line), TSB levels of 17infants selected in Lisbon
Maternity Wards are within therange considered to be susceptible of
causing bilirubin en-cephalopathy (119).
Risk Factors for Severe Jaundice
The yellowish skin discoloration characteristic of
neonataljaundice progresses in a cephalocaudal direction,
beingeasily detected in the face and sclera when TSB reaches6 mg/dL
and in the rest of the body at values O10 mg/dL (120). Although TSB
is the most widely used parameterto assess the severity of
jaundice, the range of TSB levelsresponsible for the occurrence of
pathological jaundicefrequently varies among studies and clinical
observations.The lack of consistency is justified by the fact that
the bili-rubin species responsible for brain toxicity is in fact
Bf(121,122), mostly due to its ability to easily cross theBBB
(123). For this reason, several studies argue that Bfmeasurement,
or even bilirubin/albumin ratio, are betterpredictors of BIND
(124e127). Thus, assessing the riskof developing kernicterus is a
complex subject and should
ond day of life and close to 20 or even 30 mg/dL at the 5 e6
days of life,
and still elevated at the end of the first week of life. Data
are mean values
664 Brito et al./ Archives of Medical Research 45 (2014)
660e676cally reduced rate of UCB conjugation by uridine 5
-di-phosphoglucuronosyltransferase 1A1 (116). The excretionof
bilirubin is also noticeably decreased in the neonatedue to the
poor intestinal flora that usually metabolizesUCB in excretable
products. Also, elevated b-glucuroni-dase levels, an enzyme that
deconjugates the conjugatedbilirubin species, favor UCB
reabsorption into the entero-hepatic circulation (117). Moreover,
such reabsorption ofUCB can be promoted by breastfeeding, which is
believedto increase the concentration of b-glucuronidase
(118).Consequently, TSB levels increase in the first days of
life,mainly due to UCB species, a condition that
progressivelynormalizes towards the end of the first week of life
with notreatment required and usually referred to as
physiologicaljaundice of the neonate. In a previous study performed
atour laboratory at the Faculdade de Farmacia, Universidadede
Lisboa (119), we evaluated TSB levels in 134 healthyinfants
selected by neonatologists at Hospital D. Estefa^nea(Lisbon) during
the first 3 days and in the seventh day ofrubin (TSB) may increase
dramatically and triggerbilirubin-induced neurologic dysfunction
(BIND) with mi-nor brain deficits or more severe encephalopathy
with ker-nicterus and even death (109,110) that is more frequent
ininfants presenting a TSB of at least 20 mg/dL (342 mM)(108).
Kernicterus continues to occur nowadays, even indeveloped
countries, with variable prevalence rates rangingfrom 0.6e2.7/100
000 (111). Interestingly, in low- andmiddle-income countries,
jaundice is one of the top fivecauses of neonatal death and a
significant proportion of sur-vivors manifest signs of kernicterus
(112). In 249 infantsadmitted to Cairo University Childrens
Hospital with aTSB of $25 mg/dL during a 12-month period, 44
children(14%) presented bilirubin encephalopathy (113). In thesame
study it was shown that sepsis should be considereda risk factor.
Thus, understanding the steps involved inbrain injury progression
during severe hyperbilirubinemiathat lead to the occurrence of
kernicterus is critical in orderto prevent this devastating
condition.
Bilirubin Metabolism in Perinatal Life
UCB is a tetrapyrrolic molecule formed by the catabolismof heme,
which is mostly present in erythrocyte hemoglo-bin. Due to its low
aqueous solubility, UCB has to be trans-ported in the blood bound
to a carrier protein, albumin,before conjugation in the liver. The
most neurotoxic speciesis believed to be the small fraction that
circulates as un-bound species, termed free bilirubin (Bf)
(114,115).
A series of events concerning bilirubin metabolism makejaundice
a very common occurrence in the neonatal period.Particularly,
neonates have an increased number of erythro-cytes that due to
their shorter lifespan contribute to bili-rubin production. In
addition, UCB clearance in thenewborn is less effective than in the
adult, with a dramati-life. As depicted in Figure 2 (grey line),
the average values(SD) are !10 mg/dL (171 mM) and tend to normalize
atthe seventh day of life. However, in some newborn infantsUCB
levels may increase more markedly, starting earlier or
(SD) from 134 healthy infants selected by neonatologists at
HospitalD. Estefa^nea (Lisbon) and 17 cases of infants evaluated in
Dora Brites lab-
oratory at the Faculdade de Farmacia, Universidade de Lisboa,
selected in
Lisbon Maternity Wards by showing values of TSB considered to be
sus-
ceptible of causing bilirubin encephalopathy. Data derived from
Brites D
(119).Figure 2. Total serum bilirubin (TSB) concentrations in
the first week of
life in physiological and pathological jaundice. TSB
concentrations in
the first week of life greatly differ between healthy infants
with levels usu-
ally!10 mg/dL, and normalizing at the seventh day of life
(physiologicaljaundice), and severely ill infants with TSB values
of 12 mg/dL at the sec-
th th
-
be based on the estimation of Bf, TSB and bilirubin/albu- in
both neurons and glial cells (143) as reviewed by Brites
665BloodeBrain Barrier in Neonatal Jaundicemin ratio.Neonatal
jaundice has many aggravating factors that in-
crease the risk of developing kernicterus with low TSBvalues,
including prematurity, low birth weight, and thesimultaneous
presence of other injurious conditions, suchas sepsis (128e130).
Moreover, the development of highlevels of TSB in the first hours
of life increases this riskdramatically. Nowadays, formulas based
on nomogramsmay be found in the current literature, facilitating
the deter-mination of the clinical risk of an icteric infant
(131).Recently, the plotting of transcutaneous bilirubin
measure-ment on a TSB nomogram also has been proposed as a toolfor
predictive characteristics (132), but further studies
areneeded.
Neurological Manifestations of Severe Jaundice
In the course of a severe jaundice condition there is a
pref-erential UCB brain deposition in specific regions such asthe
basal ganglia, hippocampus and cerebellum(133e136). The selective
vulnerability of these brain re-gions has been further supported by
the observation ofextensive neuronal loss and myelination defects
in autopsycases of kernicterus (133,135,137). Perlman et al.
(133)also reported the presence of brain edema in autopsy casesof
kernicteric infants, suggesting an enhanced permeabilityof the
vascular walls. Thus, it is well established that thereis a
region-specific vulnerability to UCB harmful effects,but the
underlying reasons for the preferential depositionof UCB in
selective brain regions are still unclear andshould be clarified in
order to better understand the under-lying mechanisms and to
prevent BIND.
The consequences of BIND may be either reversible
orirreversible. Some of the reversible effects include lethargyand
decreased feeding, high-pitched cry, fever, and sei-zures. On the
other hand, the irreversible sequelae are char-acterized by the
classical manifestations of choreoathetoidcerebral palsy, isolated
auditory loss and dental enamel hy-poplasia that may culminate in
death (110,138). In addition,several studies revealed that neonatal
jaundice could havean impact on learning and memory and in
long-term cogni-tive disabilities (139) and may even increase the
risk of psy-chological development outcome, especially
autisticdisorders (140).
Mechanisms of UCB Neurotoxicity
The mechanisms of UCB toxicity have been extensivelystudied and
include a general impairment of the cell mem-brane structure,
properties and function (141,142). Bili-rubin toxicity to brain
cells has been widely studied andinvolves some common features such
as the induction ofcell death (by both apoptosis and necrosis),
production ofpro-inflammatory cytokines (mainly by astrocytes and
mi-croglia) and oxidative stress, as well as specialized
features(144) and Brites and Brito (143). In fact, several
studieshave shown that elevated levels of UCB are responsiblefor
neuronal oxidative stress (145,146). UCB is able tointeract with
the mitochondrial membrane, triggering theswelling of the organelle
and the release of cytochrome c,with consequent caspase-3
activation and apoptotic celldeath (147). Interestingly, these
neuronal effects appear tobe mediated by the increased expression
of neuronal NOsynthase (nNOS) and consequent NO
production(148,149). In addition, UCB induction of oxidative
stressin the brain involves the inhibition of cytochrome c
oxidaseactivity (146,150) together with superoxide anion
radicalproduction, ATP release and disruption of glutathione
redoxstatus (146). Other neuronal effects of UCB include
neuriticatrophy and reduced neuronal arborization
(151,152).Moreover, studies on rodent neurons exposed to UCB
haveshown that the most affected neurons were those from
thehippocampus (153), and that this increased susceptibilitywas
reflected in compromised neuronal differentiation,development and
plasticity (152). Together these resultsshow the extreme
vulnerability of neurons to UCB, whichmight ultimately lead to the
impairment of the formationof neuronal circuits in the brain, thus
compromising brainfunction and eventually facilitating the
development ofneurological disorders.
UCB neurotoxicity also involves the induction of celldeath in
astrocytes, microglia and oligodendrocytes(154e156). To note that
immature astrocytes and neuronsare more susceptible to UCB-induced
cell death than matureor old cells (151,157) what may explain the
aggravatingrole of prematurity in UCB toxicity and the
susceptibilityof the neonatal period to UCB brain injury.
Astrocytes andoligodendrocytes exposed to UCB show signs of
mitochon-drial dysfunction and oxidative stress, which influence
theapoptotic process (156,158). UCB interaction with astrocytesand
microglia triggers the release of cytokines such as TNF-a, IL-1b
and IL-6 (154,155), which may initiate an inflam-matory condition
with potentiating effects on UCB damageto nerve cells. In addition
to the common effects of UCBon several glial cell types, these
studies reported specific ef-fects, especially on microglia and
oligodendrocytes. Particu-larly, microglia seem to have a first
neuroprotective effectwhen exposed to UCB by showing an increased
phagocyticability (155). This property was enhanced when
microgliawere treated with conditioned media from neurons exposedto
UCB (159). However, if exposed for long periods toUCB, microglial
cells revealed a phenotype considered tobe senescent and
dystrophic, lose their functional defensivedynamic properties and
ultimately die (155,160). Regardingoligodendrocytes, their
differentiation and myelinating ca-pacity are also compromised in
the presence of UCB (161)and may relate with the attention-deficit
disorders found ininfants who suffered from severe neonatal
hyperbilirubine-mia (162). Collectively, these studies highlight
the diverse
-
range of action of UCB regarding glial cells, which might ul-
glucose transport (168). The increased influx of glucose
666 Brito et al./ Archives of Medical Research 45 (2014)
660e676timately negatively influence the optimal environment
forneuronal function and BBB properties, thus
criticallycontributing to bilirubin encephalopathy.
BBB in Neonatal Jaundice
Initial studies by Cashore and coworkers in newborn
piglets(163,164) provided evidence for a clear BBB permeabilityto
unbound bilirubin in the first days of life. In fact, theseauthors
showed that the BBB is permeable to Bf and thatthe ratio of
bilirubin to albumin was higher in the brain thanin plasma after
infusion of bilirubin to 2-day-old piglets.They further showed that
the permeability was higher insubcortical regions than in the
cortex and that such perme-ability was no longer evident in
2-week-old animals. Theseobservations provided a basis for the
bilirubin entrance inbrain parenchyma, underlying the neurological
injury bybilirubin and for the kernicterus characteristic
regionalpattern, as well as for the increased susceptibility of
prema-ture infants. Later on, Zucker et al. (165) corroborated
thatUCB, mainly the uncharged diacid unbound species, wasable to
diffuse spontaneously through the hydrophobicmembrane core. In
addition, it was considered that a fastdeposition of Bf in the
brain may be followed by a slowerUCB passage across the intact BBB
and that the BBBdisruption may even facilitate transport of
bilirubin/albu-min into the brain (166). Despite these indications
ofBBB permeation by Bf and UCB and the widely establishedimpairment
of several brain cell types, involvement of theBBB itself in
UCB-induced neurotoxicity has been under-estimated. Here, we will
present and discuss the literatureshowing that relevant
microvascular-associated alterationsalso occur, thus adding the BBB
as an additional potentialplayer in UCB neuropathology.
Alterations of BBB Endothelium
BMEC are the first brain cells to contact with UCB in theblood
circulation, either bound to albumin or in the freeform. Thus, it
is reasonable to believe that this first contact,in addition to
providing UCB passage into the brain, mayalso be directly involved
in the pathogenesis of UCB en-cephalopathy if we consider that
endothelial response anddysfunction can be produced in such
process. Indeed, theBBB may be envisaged as having an active
participationin the pathological process resulting from severe
hyperbilir-ubinemia. Three decades ago it was described for the
firsttime that during conditions of severe
hyperbilirubinemia,binding of UCB to isolated brain capillaries may
occur(167). More recently, Cohen and collaborators (168)
furtherindicated that UCB toxicity to bovine aortic
endothelialcells involves an altered expression of GLUT-1, the
trans-porter assuring the brain uptake of glucose (4).
Moreover,expression of this transporter was enhanced, especially
inthe plasma membrane, with matching increased rate ofmay
constitute a compensatory ATP synthesis by glycolysiswhen
mitochondrial energy production is impaired, asobserved in neurons
exposed to UCB (146). Even thoughthis outcome alone disturbs the
homeostasis of the CNS,other findings suggest that high glucose
levels enhanceUCB toxicity to BMEC by generating increased
productionof reactive oxygen species (ROS) (169).
ROS modulate cerebrovascular permeability through adiverse
extent of inflammatory mediators such as NO andcytokines (170,171).
In our own studies using primary cul-tures and a cell line of human
BMEC (HBMEC) as a simplemodel of the BBB (172,173) exposed to
UCB/human serumalbumin ratios (UCB/HSA) mimicking moderate (UCB/HSA
5 0.5) and severe (UCB/HSA 5 1.0), we have shownthat UCB disturbs
endothelial homeostasis (174). In fact,the interaction of UCB with
HBMEC induced a fast andsustained elevation of eNOS expression,
followed by amarked accumulation of nitrites, a widely used
biomarkerof NO production (Figure 3A). These results indicate
thatHBMEC are exposed to nitrosative stress to which mayaccount the
sustained disruption of glutathione metabolismas revealed by the
elevated ratio of oxidized glutathione(GSSG) relative to total
glutathione (GSx), also suggestingoxidative stress (Figure 3A). UCB
additionally affected thesecretion of cytokines as depicted in
Figure 3B. In fact, ashort exposure to UCB inhibited the release of
interleukin(IL)-6, IL-8 and VEGF, which is followed by an
increasedrelease, first of IL-6 already at 4 h, followed by IL-8
andlater on by VEGF (174). Interestingly, a similar effectwas
observed for IL-6 secretion by astrocytes, with an earlyinhibition
followed by the cytokine upregulation by moreprolonged exposure to
UCB (154). Overall, these eventsshow that UCB is able to trigger an
endothelial injuriousresponse with the production of important
mediators thatmay aggravate the inflammatory condition in the brain
pa-renchyma or even impact the endothelial integrity itself
byacting in an autocrine manner.
eNOS, VEGF and VEGFR-2 may interact with caveolin-1 (24e26).
Caveolin-1, the major structural protein of cav-eolae as reported
above, not only ensures the movement ofsubstances via transcytosis
but also has an active role in theregulation of BBB vesicular
permeability (20,22).Caveolin-1 is also involved in various aspects
of signaltransduction pathways and has been associated
withincreased BBB permeability and brain edema during injury(23).
Interestingly, UCB caused an increase in the levels ofcaveolin-1
(Figure 4A), as well as in the number of caveo-lae (Figure 4B),
reflecting an augmented intracellular trans-port and permeability
(175).
Caveolin-1 is known to interact with the ABC trans-porter P-gp
(24e26). This efflux transporter has shown animportant role in the
transport of UCB across the endothe-lial monolayer through a
significant active efflux of thismolecule in the basolateral to
apical direction (176). These
-
observations are corroborated by the disrupted junctions and
n of c
C wi
e exp
levat
paire
on, fo
r (VE
667BloodeBrain Barrier in Neonatal Jaundicedata support the
notion of UCB as a substrate of effluxtransporters such as P-gp
(177,178). Interestingly, recentdata from our group showed that
BMEC exposed to UCBpresent an inhibition of P-gp activity (179).
This observa-tion may justify the increased expression of the
proteinobserved in human brain tissue samples obtained from
akernicterus case (136), probably as a compensatory mecha-nism
created by the brain to overcome the decreased activ-ity and to
maintain the efflux of UCB.
ROS can regulate the transcription of MMPs. These areproteolytic
enzymes with the ability to degrade all types of
Figure 3. Unconjugated bilirubin (UCB) interferes with the
secretion patter
thelial cells (HBMEC). Data ensued from treatment of a cell line
of HBME
mimics a severe neonatal jaundice, for the indicated time
points. Results wer
expression of endothelial nitric oxide synthase (eNOS), as
indicated by the e
oxide (NO). In parallel, the cell defense system provided by
glutathione is im
total glutathione (GSx). There is also an early inhibition of
cytokines secreti
to be released, followed by IL-8, whereas vascular endothelial
growth factoECM proteins, thus participating in tissue
regeneration, re-modeling or even angiogenesis (180). However, in
patholog-ical conditions MMPs may lead to ECM degradation
andweakened anchoring of the endothelium to the basementmembrane
(181). This may occur in cases of severe jaundiceonce we
demonstrated that UCB leads to MMP-2 and -9 acti-vation in the
extracellular media of treated BMEC (179).
Increased levels of UCB may then lead to HBMEC reac-tivity that
ultimately can result in BBB impairment, facili-tating UCB passage
into the brain. Together it appears thatthe UCB-induced expression
and release of NO and cytokinesaffects the endothelial cells as a
stable barrier. This contributesto alterations in AJ and TJ
proteins (175,179) as observed inFigure 4C for the TJ protein ZO-1,
leading to weakness ofthe TJ strands and to the retraction of
cell-to-cell contacts(Figure 4D and E). In accordance, UCB caused a
decreasein transendothelial electrical resistance (TEER) and an
in-crease in paracellular permeability to the low molecularweight
compound sodium fluorescein (376 Da) (175). Theseare widely used
indicators of barrier integrity (4), reinforcingthat the compromise
of the BBB integrity may be one of thecompromised barrier integrity
obtained in rat BMEC (179)and intestinal epithelial cells
(182).
Ultimately, the damage of UCB to HBMEC lead to the in-duction of
cell death by apoptosis and necrosis (174,179,183),towhichmay
account the increased cellmembrane fragility byUCB, as suggested by
the release of cellular fragments andeven entire cells from the
monolayer (175).
Impairment of Pericytesevents additionally contributing to UCB
neurotoxicity. These
ytokines and disrupts the redox status of human brain
microvascular endo-
th 100 mM UCB in the presence of 100 mM human serum albumin,
which
ressed as fold change from the respective controls. UCB
stimulates an early
ed expression at 1 h, which is followed by an increased
production of nitric
d, as shown by the elevation of the ratio of oxidized
glutathione (GSSG) to
llowed by their later increased production. Interleukin (IL)-6
is the first one
GF) presents a delayed response. Data derived from Palmela et
al. (174).Brain microvascular pericytes extensively enwrap
capil-laries, sharing basement membrane with BMEC and influ-encing
their properties (7). So, pericyte deficiency in theCNS contributes
to BBB breakdown and brain hypoperfu-sion resulting in
neurodegenerative changes (184). A paucityof pericytes in germinal
matrix vasculature of premature in-fants has been suggested to
contribute to hemorrhage pro-pensity (48). Despite the relevance
that pericytes may havein the mechanisms of BBB disruption by UCB,
no informa-tion in addition to what is presented here is
available.
Our recent and unpublished studies showed that UCB inconditions
mimicking a moderate and severe hyperbilirubi-nemia induces a rapid
increase in eNOS expression byhuman brain vascular pericytes
(HBVP), already noticedat 1-h incubation (Figure 5A). Such effect
may be behindthe increase in NO production by UCB, which was
noto-rious (|4-fold increase) at 72 h after UCB addition(Figure
5B). As already mentioned, NO is among the stim-uli that influence
endothelial properties (171). Therefore,the release of NO by HBVP
adds to that produced by
-
668 Brito et al./ Archives of Medical Research 45 (2014)
660e676BMEC, reinforcing the homeostasis disruption. Moreover,it
may impact on neurons where NO contributes to celldeath as
demonstrated in experiments where the NOS in-hibitor L-NAME
prevented neuronal death (148).
Figure 4. Unconjugated bilirubin (UCB) induces caveolae
formation and disrupt
Data ensued from treatment of a cell line of HBMEC with 50 or
100 mMUCB in t
a severe neonatal jaundice, respectively. UCB induces the
expression of caveolin-1
(arrows) as shown by transmission electron microscopy (B). It
also decreases th
shown by fluorescence microscopy (C), decreases the tight
junction strands (arr
the intercellular spaces (arrows), as shown by scanning electron
microscopy (E)UCB also induced the secretion of cytokines by
HBVP.Indeed, the expression of IL-6 mRNAwas upregulated at 45min
and that of VEGF mRNA at 4 h, with the subsequentcytokine
secretion, already marked at 1 h for IL-6 and
s tight junctions in human brain microvascular endothelial cells
(HBMEC).
he presence of 100 mM human serum albumin, which mimic a
moderate and
as observed by fluorescence microscopy (A) and the formation of
caveolae
e expression of the tight junction protein zonula-occludens-1
(arrows) as
ows) visualized by freeze-fracture electron microscopy (D), and
increases
. Adapted from Palmela et al. (175) with permission.
-
669BloodeBrain Barrier in Neonatal Jaundicepeaking at 24 h for
VEGF (Figure 5C and D). It is inter-esting to point out that the
HBVP response to UCB occursfaster than that observed for BMEC
(Figure 3B) where IL-6secretion was only detected at 4 h and that
of VEGF at 72 h
Figure 5. Unconjugated bilirubin (UCB) induces nitrosative
stress, a pro-inflamm
cytes (HBVP). Data ensued from treatment of primary cultures of
HBVP with 50
mimics a moderate and a severe neonatal jaundice, respectively,
for the indicated
synthase, as indicated by the elevated expression at 1 h (A),
which is followed b
There is also upregulation of interleukin (IL)-6 and vascular
endothelial growth fa
(D). A loss of HBVP was observed by phase contrast microscopy
(E). Nuclear fea
Hoechst 33258 dye (F) and the percentage of apoptotic cells was
quantified (G)(174). Also interesting is the fact that the HBVP
release ofIL-6 even precedes the one by astrocytes and
microgliatreated with UCB (154,155). Therefore, secretion of
thesemediators by HBVP may further contribute to the injurious
atory response and compromises the viability of human brain
vascular peri-
or 100 mM UCB in the presence of 100 mM human serum albumin,
which
time points. UCB stimulates an early expression of endothelial
nitric oxide
y an increased production of nitrites (B), the end product of
nitric oxide.
ctor (VEGF) mRNA (C), followed by the corresponding cytokines
secretion
tures of apoptosis (arrow) were observed by fluorescence
microscopy with
. *p !0.05 and **p !0.01 vs. respective control.
-
670 Brito et al./ Archives of Medical Research 45 (2014)
660e676effects by directly promoting BBB disruption, to whichshall
also account the proper death of pericytes. Indeed, atime- and
concentration-dependent loss of cell viabilitywas visualized by
phase contrast microscopy (Figure 5E),and apoptotic features of
apoptosis were significantlyenhanced (Figure 5F and G).
Collectively, data revealed impairment of HBVP byUCB in relevant
physiopathological conditions. Due tothe importance of these cells
in CNS homeostasis, futurestudies should explore the mechanisms
involved in pericytedemise and their reflex in the NVU compromise
in order todevise ways to modulate them.
Compromise of the NVU
One of the most interesting findings obtained in the in
vitrostudies was that several players that mediate the
formation
Figure 6. The brain parenchyma of a kernicterus case presents
vascular and neu
topsied premature neonate with kernicterus and in an age-matched
control with
marker cluster of differentiation (CD) 34 shows an increase in
microvascular den
of immature vessels (inserts). (B) A poor Luxol-Periodic Acid
Schiff (Luxol-PA
Decreased neurofilament immunoreactivity shows the impairment of
neuritic arb
permission of Journal of Child Neurology.of new vessels were
modified by UCB. Particularly, theinteraction of UCB with HBMEC
revealed to induce anincreased production of IL-6, NO and VEGF,
upregulatedlevels of VEGFR-2 and augmented endothelial
perme-ability (174,175), parameters that have been implicated inthe
angiogenic process (185e190). In accordance withthese evidences,
our studies on human neonatal brain sam-ples from autopsy material
of a kernicterus case revealed amarked increase in vessel density
in the cerebellum (137)(Figure 6A). Such effect was also observed
in the corpusstriatum, in the brainstem and in the hippocampus
(136).Interestingly, the microvessels presented a poorly
definedlumen, a characteristic of immature and
hyperpermeablevessels. This observation is in line with the data
obtainedin our in vitro studies with HBMEC, suggesting an
angio-genic process that might function to compensate
theUCB-induced dysfunction.
ronal alterations. Immunohistochemical and histological analysis
in an au-
out brain pathology. (A) Immunohistochemical analysis of the
endothelial
sity, and the presence of vessels with a poorly defined lumen,
characteristic
S) staining in the kernicterus case indicates a loss of myelin
fibers. (C)
orization in the kernicterus case. Reproduced from Brito et al.
(137) with
-
Curiously, the kernicteric brain showed an increased as
prematurity, hypoxia and sepsis, making difficult the
the detachment of endothelial cells from the monolayer,
whichconstitute signs of barrier fragility. UCB damage is extended
to
Together theBBBappears as a possibleCNSplayer tomodulate
7. Sa-Pereira I, Brites D, Brito MA. Neurovascular unit: a focus
on peri-
671BloodeBrain Barrier in Neonatal Jaundiceexpression of VEGF in
cerebellar neurons (137), remainingto clarify whether these cells
are the producers or the maintargets of the vascular permeability
factor. The accumulationof VEGF in Purkinje neurons may underline
the preferentialpoisoning effect of bilirubin to such neurons
already sug-gested (191) and contribute to the abnormal tone and
motorcoordination in children with kernicterus (110).
Upregulationof VEGF was paralleled by an increased expression
ofVEGFR-2 (137), suggesting an autocrine/paracrine effectof UCB on
neurons that can perpetuate homeostatic pertur-bations. The
presence of the blood component albumin inthe brain parenchyma was
also noticed (136), pointing to mi-crovessel leakage that was
previously reported to be respon-sible for neurotoxic
manifestations (192e194).
Vascular alterations in the kernicterus case were accom-panied
by neuronal dysfunction, characterized by the lossof myelin fibers
(Figure 6B). In addition, there was animpairment of the dendritic
arborization in the cerebellum,and particularly in the Purkinje
neurons (Figure 6C),corroborating both in vitro findings (151,161)
and thosereported in a mouse model of hyperbilirubinemiawhere loss
of Purkinje neurons was observed (195). Dam-age is not restricted
to the cerebellum, and impairment ofthe basal ganglia was a common
feature in otherhuman neonatal brain samples from infants with
kernic-terus (196).
Increased expression of the efflux transporters
multidrugresistance-associated proptein 1 (MRP1) and P-gpdescribed
in the kernicterus case (136,137) is in line witha compensatory
mechanism of protection against UCBneurotoxicity (197). Indeed, the
inhibition of MRP-1 byMK571 enhanced UCB neurotoxic effects (198).
A signif-icant upregulation of P-gp expression in microvessels
ofthe jj (jaundiced) Gunn rats (a kernicterus model) ascompared
with Jj (not jaundiced) littermates additionallyhighlights the
relevance that such efflux transporter mayhave on bilirubin
encephalopathy (199). Furthermore, ifwe consider that the
expression of P-gp increases untiladulthood (15,199), we may
speculate that the blood ves-sels of the mouse pups have a low
capacity to accomplishthe efflux UCB from the brain parenchyma.
CompromisedNVU in neonatal life may similarly result from
thedecreased expression of MRP1 previously observed inimmature
neurons in primary culture (198).
Collectively, upregulation of the efflux transporters
mayrepresent an attempt to protect the brain from UCBentrance, but
the leakiness of the microvasculature clearlyseems to counteract
such process. Collected data are impor-tant to open new avenues for
future research aiming tobetter understand whether vascular
dysfunction is a deter-minant or a secondary event in bilirubin
encephalopathy.Added to that, the evaluation of additional cases of
kernic-terus is fundamental. These cases frequently occur ininfants
who present concomitant risk factors such cytes. Mol Neurobiol
2012;45:327e347.whenever brain dysfunction by lasting
hyperbilirubinemia issuspected in order to prevent neurological
deficits by UCB.
AcknowledgmentsWe acknowledge our funding support from Fundac~ao
para aCie^ncia e a Tecnologia (FCT), Lisbon, Portugal, through
grantsPTDC/SAU-NEU/64385/2006 (to DB), PTDC/SAU-FCF/68819/2006 (to
MAB), and the strategic project PEst-OE/SAU/UI4013/2011e2014, as
well as FEDER.
Conflicts of interest: The authors declare no conflict of
interest.
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