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Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. The Blood-Brain Barrier: New Research : New Research, edited by Pedro A. Montenegro, and Stefanee M. Juárez, Nova Science Publishers,

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. The Blood-Brain Barrier: New Research : New Research, edited by Pedro A. Montenegro, and Stefanee M. Juárez, Nova Science Publishers,

NEUROSCIENCE RESEARCH PROGRESS

THE BLOOD-BRAIN BARRIER

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

NEW RESEARCH

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The Blood-Brain Barrier: New Research : New Research, edited by Pedro A. Montenegro, and Stefanee M. Juárez, Nova Science Publishers,

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The Blood-Brain Barrier: New Research : New Research, edited by Pedro A. Montenegro, and Stefanee M. Juárez, Nova Science Publishers,

NEUROSCIENCE RESEARCH PROGRESS

THE BLOOD-BRAIN BARRIER NEW RESEARCH

PEDRO A. MONTENEGRO AND Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

STEFANEE M. JUÁREZ EDITORS

Nova Science Publishers, Inc. New York The Blood-Brain Barrier: New Research : New Research, edited by Pedro A. Montenegro, and Stefanee M. Juárez, Nova Science Publishers,

Copyright © 2012 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Library of Congress Cataloging-in-Publication Data The blood-brain barrier : new research / editors, Pedro A. Montenegro and Stefanee M. Juarez. p. ; cm. Includes bibliographical references and index. ISBN:  (eBook) I. Montenegro, Pedro A. II. Juarez, Stefanee M. [DNLM: 1. Blood-Brain Barrier. WL 200] 573.8621--dc23 2011038570

Published by Nova Science Publishers, Inc. † New York The Blood-Brain Barrier: New Research : New Research, edited by Pedro A. Montenegro, and Stefanee M. Juárez, Nova Science Publishers,

CONTENTS Preface Chapter I

Chapter II

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Chapter III

Chapter IV

Chapter V

Chapter VI

vii Local and Temporal Regulation of the Blood-Brain Barrier during Normal and Altered Physiological States Beatriz Gómez-González, Gabriela Hurtado-Alvarado and Javier Velázquez-Moctezuma Angiogenesis and its Mechanistic Implications in the Pathology of Neurodegenerative Disorders Aditiben Patel, Giuseppe V. Toia, Bill Hendey and Paul M. Carvey It Takes Two to Tango: Protein-protein Interactions in the Translocation of Pathogens across a Blood-brain Barrier L. Pulzova, P. Mlynarcik, E. Bencurova and M. Bhide Efflux-Transporters at the Blood-Brain Barrier: Therapeutic Opportunities Alan Talevi and Luis Enrique Bruno-Blanch Novel Strategies to Restore Blood-Brain Barrier Integrity after Brain Injury Winfried Neuhaus, Malgorzata Burek, Christian Wunder and Carola Y. Förster Evaluation of the Blood-Cerebrospinal Fluid Barrier in Neurological Diseases Alina González-Quevedo, Rebeca Fernández Carriera, Sergio González García and Idalmis Suárez Luis

1

43

79

117

145

173

Chapter VII

Blood-Brain Barrier in Health and Disease Inês Palmela, Dora Brites and Maria Alexandra Brito

201

Chapter VIII

Cell Culture Models of the Blood-Brain Barrier: New Research Nicola F. Fletcher and John J. Callanan

219

The Blood-Brain Barrier: New Research : New Research, edited by Pedro A. Montenegro, and Stefanee M. Juárez, Nova Science Publishers,

vi Chapter IX

Contents HIV-1 Gp120 Induces Blood-Brain Barrier Abnormalities: Pathophysiology and Therapeutic Consequences Jean-Pierre Louboutin and David S. Strayer

241

Chapter X

Blood Brain Barrier in Hepatic Encephalopathy Cuiming Sun and Pei Liu

259

Chapter XI

Blood-Brain Barrier (BBB): Morphology and Disease L. Colin-Barenque, A. Zepeda-Rodriguez, R. Jimenez-Martinez, A. Gonzalez-Villalva, M. Rojas-Lemus, P. Bizarro-Nevares, V. Rodriguez-Lara, F. Pasos-Najera, V. Guarner-Lans, A. Santamaria and T. I. Fortoul

271

Chapter XII

Role of Blood-Brain Barrier in Cerebral Malaria Mauro Prato

287

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Index

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313

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PREFACE The blood-brain barrier (BBB) is a highly regulated system that maintains brain allostasis. BBB achieves its main function by transporting blood to brain glucose, amino acids and other molecules needed for proper neural physiology, while extruding from the brain molecules derived from neural and glial metabolism that may have neurotoxic properties. In this book, the authors present topical research in the study of blood-brain barriers including their local and temporal regulation during normal and altered physiological states; the therapeutic opportunities of efflux-transporters at the blood-brain barrier; novel strategies to restore blood-brain barrier integrity after brain injury; evaluation of the blood-cerebrospinal fluid barrier in neurological diseases and blood-brain barrier in hepatic encephalopathy. Chapter 1 - The blood-brain barrier (BBB) is a highly regulated system that maintains brain allostasis. BBB achieves its main function by transporting from blood to brain glucose, amino acids and other molecules needed for proper neural physiology, while extruding from the brain molecules derived from neural and glial metabolism that may have neurotoxic properties. BBB function is tightly regulated by local synaptic and glial activity; both nerve and glial cells release molecules that modify hemodynamic and permeability parameters at the BBB. Under normal physiological conditions, products released by neural activity, such as some neurotransmitter/neuromodulator molecules and byproducts of neural metabolism, exert vasodilator effects and induce changes in BBB permeability. Active neurons have been shown to release several molecules that quickly increase cerebral blood flow locally, with a concomitant rise in BBB permeability to glucose and other solutes. In order to achieve prolonged activity-related effects on cerebral blood-flow and vessel permeability astrocytes are needed. Astrocyte calcium waves induce the release of vasoactive molecules that exert long-term hemodynamic and permeability changes in local vessels. Under altered physiological conditions, BBB function is also modified in a brain region specific manner. The mediators of the changes are almost the same as those of the normal physiological state, but their effects are intensenly and prolonged. Stress, a state of disrupted allostasis, has been shown to increase BBB permeability to blood-borne potentially neurotoxic molecules through increased vesicle-mediated transport at adulthood and during early-life; the effects are observed in some cortical regions, the brain stem, cerebellum, hippocampus, basal ganglia, and cervical spinal cord. Both adult and early-life stress exerted its effects upon BBB through increased release of serotonin, corticotrophin-releasing hormone, and pro-inflammatory cytokines.

The Blood-Brain Barrier: New Research : New Research, edited by Pedro A. Montenegro, and Stefanee M. Juárez, Nova Science Publishers,

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Pedro A. Montenegro and Stefanee M. Juárez

Sleep restriction is another altered physiological state that is accompanied by increased BBB permeability to blood-borne potentially toxic molecules. In a rat model, sleep restriction to 4-hours per day increased BBB permeability to an albumin-bound dye in almost the whole brain; the mechanism of the increased vessel permeability may be sleep deprivation-related hyperthermia, increased pro-inflammatory cytokine production and maybe some byproduct of altered neural and glial function after chronic sleep restriction. In conclusion, BBB function is tightly regulated inclusive during altered physiological states, some brain regions are affected during stress exposure or sleep restriction but others are spared and have normal brain vessel permeability. More research is needed to clarify regional differences in BBB properties and to elucidate the temporal organization of the involved molecules. Chapter 2 - Multiple Sclerosis, Alzheimer’s disease, Parkinson’s disease, Amyotrophic Lateral Sclerosis, Huntington’s disease and Neuro-AIDS are progressive neurodegenerative diseases (NDs) with disparate symptomatic characteristics associated with degenerative changes in differing parts of the brain. Yet, these NDs are all associated with neuroinflammation. An emerging literature suggests that the shared neuroinflammation of these NDs is also associated with a dysfunctional blood brain barrier (BBB) implicating a currently underappreciated role for the periphery in their etiopathogenesis and potentially, their progressive nature. Although neuroinflammatory mediators can directly and adversely affect BBB function, several of these mediators are also pro-angiogenic. Thus, angiogenesis is likely a compensatory response to neuroinflammation. Moreover, developing vessels are inherently leaky while chronic angiogenesis can lead to aberrant vessel formation and a similarly dysfunctional BBB. This review will argue that the compensatory angiogenesis that accompanies neuroinflammation likely leads to a dysfunctional BBB that would facilitate the entry of peripheral toxins and elements of adaptive immunity that may well contribute to disease progression. If indeed angiogenic changes, and chronic angiogenesis in particular, compromise BBB function and contribute to disease progression, then anti-angiogenic drugs that are currently in clinical trials or already FDA approved may stabilize barrier dysfunction and slow disease progression. If this were true, then anti-angiogenic drugs may potentially slow or even halt the progression of several NDs advocating exploratory clinical trials to test this hypothesis. Chapter 3 - Blood-brain barrier (BBB) is a regulatory interface between the peripheral circulation and the central nervous system (CNS), which has unique role in the protection of the brain from toxic substances and pathogens present in the blood. Many pathogens including parasites, bacteria, viruses and fungi have the potential to infect the CNS, but it is unclear why only a relatively small number of pathogens account for the most clinical cases with nervous disorders. Pathogens may enter the CNS via transcellular penetration, paracellular passage and/or via “Trojan horse” mechanism (via infected phagocytes). Interactions between protein molecules from host and pathogens are crucial to trigger translocation processes. Indeed, it takes two to tango: both host receptors and pathogen ligands. Underlying molecular basis of BBB translocation by various pathogens has been revealed in the last decade, however, an array of protein-protein interactions between many of the neuroinvasive pathogens and BBB remained fully unexplored. Identification and molecular characterization of these pathogens and host factors mediating BBB penetration can open novel ways and perspectives in the development of more specific drugs and

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Preface

ix

vaccines strategies. This chapter will give new insight into the various protein-protein interactions that take place in BBB translocations process. Chapter 4 - ABC efflux transporters are ATP-dependent carriers responsible for regulating the entry of endogenous and exogenous compounds to certain body compartments and facilitating their elimination. Thus, they are especially expressed in barrier tissues (e.g. gut walls o blood-brain barrier) and in organs implicated in elimination functions (e.g. liver and kidneys). ABC transporters are characterized by wide-substrate specificity and they have been associated to multi-drug resistance issues. The level of expression of these transporters at the blood-brain barrier determines (at least partially) the inability of drugs from several therapeutic categories to reach their molecular targets in therapeutically effective concentrations. Here the authors will overview recent reports on the expression, localization and regulation of ABC transporters at the blood-brain barrier, and their association to certain disorders: inflammation, epilepsy, brain cancer, Alzheimer’s disease. The authors will describe the therapeutic opportunities derived from these studies: development of ABC transporters inhibitors/modulators; modeling of novel drugs capable of avoiding recognition by efflux transporters, and; carrier systems designed to circumvent the biochemical component of the blood-brain barrier. Chapter 5 - Ischemic brain damage concerns very different diseases and conditions, which might be acute, sub-acute conditions or long-term outcome. Ischemic brain disorders (stroke, traumatic brain injury, ischemia/reperfusion, hemorrhage, infarction) share several common features, one of which is a disruption of blood-brain barrier (BBB) integrity in the area of injury and its penumbra. The BBB accounts for the low extracellular concentrations of amino acids and proteins in the brain relative to the blood, and also restricts access to the immune system and of systemically administered hydrophilic drugs and neuroactive substances. The main structures constituting the barrier are the tight junctions sealing the microvascular endothelial cells of the blood-brain barrier. BBB forming brain capillary endothelial cells express the TJ proteins occludin and claudin-5 and claudin-12. Specific transport proteins expressed on cells of the barrier allow for active transport of metabolic products such as glucose across the barrier. When the BBB becomes disrupted as in acute or post-acute states of ischemic brain damage, vasogenic edema occurs causing a net flux of water from blood to the brain tissue. Cerebral ischemia produces cytotoxic oedema initially, followed by vasogenic edema when the vascular endothelial function is severely altered. The clinical strategies thus aim to limit the development of edema and to create the best conditions for adequate perfusion of brain parenchyma to avoid ischemia. In this chapter the authors will summarize and review the most current strategies to restore blood-brain barrier integrity to treat and limit ischemia and edema formation following brain damage, in order to alleviate acute conditions and help counteract long-term neurological damage. Chapter 6 - The brain functions within a well-controlled environment that must be separated from the general internal environment of the body. The mechanisms that control the composition of this milieu are unified in a term conveniently designated as the “blood brain barrier” (BBB). The anatomical basis of this barrier lies in the intercellular junctions between cells forming the interface between blood and brain (BBB) and in the choroid plexuses (blood–cerebrospinal fluid [CSF] barrier). These cell junctions (tight junctions) occlude, or at least severely attenuate, movement through intercellular spaces between endothelial cells in the blood–brain barrier and epithelial cells in the choroid plexus blood–CSF barrier,

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Pedro A. Montenegro and Stefanee M. Juárez

transforming the properties of the individual endothelial and epithelial cells into properties of these interfaces as a whole. But the BBB is much more than just a barrier made up of tight junctions, it is a metabolically active, regulated, and regulatory interface, with transport, secretory, and enzymatic activities. The BBB that resides within the endothelial cells of the cerebral capillaries, the bloodcerebrospinal fluid barrier (B-CSFB) and the meninges function together to control the internal environment of the brain and the spinal cord. The B-CSFB constitutes another wellknown anatomical barrier which exists between the blood and the cerebrospinal fluid (CSF). Differing from cerebral capillaries, the microvessels in the choroid plexus, are fenestrated and do not establish any barrier function, while the anatomical site of the B-CSFB is located within the epithelial cells of the choroid plexus that are closely joined with tight junctions Chapter 7 - The blood-brain barrier (BBB) is an elaborate structure of utmost importance, as it separates the brain compartment and the peripheral organs, allowing the creation of a unique brain environment for optimal neuronal activity. Endothelial cells of brain microvasculature present special properties that restrict the free movement of molecules and are thus considered as the anatomic basis of the BBB. The presence of both tight and adherens junctions, together with transporters and intracellular signaling pathways, actively influence and regulate the barrier properties of these endothelial cells. Moreover, endothelial interactions with other brain cells and matrix components are crucial for the maintenance of a proper BBB. This has led to the concept of neurovascular unit, which embraces the known and crucial interactions between endothelial cells and astrocytes, pericytes and the basement membrane, as well as the possible interplay with neurons and microglia. Additionally, the endothelial cells act as a signaling interface between the compartments that are separated, being directly activated by signals from both environments and responding accordingly, therefore actively participating in brain protection. Although the role of the BBB in the progression or even as a cause of central nervous system (CNS) disorders has often been dismissed, it is nowadays believed that BBB dysfunction plays an important role in the progression of numerous CNS disorders. In fact, it has recently been documented that disruption of tight junctions occurs in multiple sclerosis, sepsis, human immunodeficiency virus (HIV) infection and brain tumours, or even in diseases like Alzheimer’s disease, Parkinson’s disease and epilepsy, in which there is also evidence of transporter deregulation. A dysfunctional or even disrupted BBB causes a perturbation of blood-brain homeostasis, with an associated increased infiltration of immune cells into the brain compartment. These events are ultimately responsible for the exacerbation of brain injury and are thus key players in disease aggravation. In this chapter, the unique properties of the BBB are explored in order to better understand the role of this complex structure in brain homeostasis. Furthermore, it is intended to increase the awareness of the importance of studying this dynamic structure, as it can be an important player in the progression of CNS disorders. Chapter 8 - The blood-brain barrier (BBB) functions as an interface between the blood and the brain parenchyma, and restricts the passage of large and hydrophilic substances into the central nervous system. In vertebrates, tight junctions between brain capillary endothelial cells form this barrier, and this phenotype is maintained by the close association of astrocytes and pericytes with brain endothelial cells. Cell culture models of the BBB have been developed to study drug and nanoparticle transport to the brain and the mechanisms used by pathogens to cross the BBB. Recently, it has been reported that pericytes play a role in the maintenance of BBB integrity, and culture models have been developed which more closely

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Preface

xi

mimic the in vivo situation by including pericytes and by culturing endothelial cells under flow conditions, which increases tight junction integrity. This review will explore new developments in in vitro BBB culture techniques, and the advances in the authors’ understanding of BBB interactions with drugs and pathogens. Chapter 9 - Many systemic and CNS diseases can compromise the blood-brain barrier (BBB). BBB disruption mediates some of the tissue damage that accompanies HIV-1 infection of the brain, and so facilitates viral entry into the CNS. Although damage to the BBB has been documented in patients with HIV-related encephalopathy, little is known about the mechanism by which this injury occurs. The authors used animal models of HIVassociated neurocognitive disorder (HAND) to characterize abnormalities of the BBB in this context. they studied BBB disruption caused by HIV-1 envelope glycoprotein 120 (gp120) as a model, and tested the protective effectiveness of antioxidant gene delivery. Exposure to gp120, whether acute (by direct intra-caudate-putamen (CP) injection) or chronic (using SV(gp120), an experimental model of ongoing production of gp120) disrupted the BBB, and led to leakage of vascular contents into the area of gp120 exposure. Gp120 was directly toxic to brain endothelial cells and gp120-mediated BBB abnormalities were related to lesions of brain microvessels. Abnormalities of the BBB may reflect the activity of proteolytic enzymes, particularly matrix metalloproteinases (MMPs). These target laminin, a major BBB component, and attack the tight junctions between endothelial cells and BBB basal laminae. MMP-2 and MMP-9 were upregulated following intra-CP gp120-injection. Gp120 greatly reduced total CP content of laminin and tight junction proteins. Reactive oxygen species (ROS) have been reported to activate MMPs. Injecting gp120 induced lipid peroxidation. One product of gp120-triggered lipid peroxidation, hydroxynonenal (HNE) was immunolocalized to vascular endothelial cells. Moreover, gene transfer of antioxidant enzymes using recombinant SV40-derived vectors protected against gp120-induced BBB abnormalities. BBB injury has been linked to NMDA, which upregulates the proform of MMP-9 and increases MMP-9 gelatinase activity. Using the NMDA receptor (NMDAR-1) inhibitor, memantine, the authors observed partial protection from gp120-induced BBB injury. Thus, 1) HIV-envelope gp120 disrupts the BBB; 2) this occurs via lesions in brain microvessels, MMP activation and degradation of vascular basement membrane and vascular tight junctions; 3) NMDAR-1 activation plays a role in this BBB injury; and 4) antioxidant gene delivery using rSV40 vectors as well as NMDAR-1 antagonists may protect the BBB. Chapter 10 - In liver failure, hepatic encephalopathy is one of the most important clinical manifestations due to hepatic dysfunction and proto-systemic shunting of the intestinal blood. Brain edema is the most serious anatomical lesion in hepatic encephalopathy. In most autopsies of patients dying from fulminate hepatitis, little alteration in the brain was observed except edema. Ammonia levels have been found to be increased in most patients in hepatic coma, however, the role of blood ammonia in the evaluation of hepatic encephalopathy is disputed. The authors’ study, consistent with other studies, observed the disrupted blood brain barrier in hepatic failure indicating disruption of BBB contributes to the brain edema as well. With the tight junction and adherent junction, BBB forms a physical barrier and regulates the brain homeostasis. Studies on the integrity of BBB in ALF show discrepant results. Some studies observed swelling of astroglial foot process with BBB remaining intact, while more recent studies, including the authors’ study, indicated the disruption of BBB contributed to the brain edema in hepatic encephalopathy. their study showed the increased permeability

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Pedro A. Montenegro and Stefanee M. Juárez

might be because of the breakdown of tight junctions and the loss of the TJ-associated occudin, and in such process the proinflammatory cytokine TNF a plays a crucial role. Chapter 11 - Some restricted areas in the body are protected by special structures identified as blood barriers; some examples are: the thymus, the retina, testes, and the thyroid gland. These structures restrict the contact of these organs with the environment selecting the nutrients and other components from the plasma. The brain is part of these selected organs with blood barrier, which is assembled by series of tight junctions with continuous capillaries. Other components of this barrier are the basal lamina, the pericytes and astrocytes feet. There are two barrier systems in the brain, the one located between general circulation and the brain tissue and the one in the choroid plexus. They are different; continuous capillaries that select nutrients from the general circulation in the first case, and in the second by fenestrated capillaries with less selectivity. Because its location and structure, both barriers could be evaluated by morphological techniques to identify their changes in experimental models or in autopsy samples. Histology, Transmission and Scanning Electron Microscopy (TEM and SEM) are excellent tools to evaluate them. This chapter will display information about the normal structures of these barriers and the changes found after the exposure to inhaled pollutants as well as in some human pathology. Chapter 12 - Cerebral malaria (CM) is the most severe neurological complication of Plasmodium falciparum infection and is associated with significant mortality or neurological sequelae in surviving patients. The pathogenesis of CM remains unclear. Since the parasite is sequestered in the vascular space without entering the brain parenchyma, it is not well understood how it is capable of inducing such a devastating neurological disease. According to the “permeability hypothesis” by Maegraith and Fletcher, which was then improved by Clark’s “cytokine theory,” a leaky blood-brain barrier (BBB) allows host toxic compounds produced in response to infection to enter the brain and cause neurological dysfunctions. However, although in animal models the BBB breakdown is clearly evident, in humans the BBB is mildly impaired only. The present chapter will review the available evidence from in vitro, in vivo and post-mortem studies in order to highlight the role of BBB damage in CM, and data on altered structural and functional integrity of the BBB will be discussed. A dedicated section will explore thoroughly the possibly crucial role of matrix metalloproteinases (MMPs), whose involvement in CM has been recently proposed. Furthermore, few examples of new drugs, including MMP inhibitors and anti-inflammatory drugs, which might block the BBB leakage and prevent CM neurological symptoms will be given. Taken altogether these data might help to better understand the role of the BBB in CM and hopefully may be useful in order to design a specifically targeted adjuvant therapy.

The Blood-Brain Barrier: New Research : New Research, edited by Pedro A. Montenegro, and Stefanee M. Juárez, Nova Science Publishers,

In: The Blood-Brain Barrier: New Research ISBN: 978-1-62100-766-1 Editors: Pedro A. Montenegro and Stefanee M. Juárez © 2012 Nova Science Publishers, Inc.

Chapter I

LOCAL AND TEMPORAL REGULATION OF THE BLOOD-BRAIN BARRIER DURING NORMAL AND ALTERED PHYSIOLOGICAL STATES Beatriz Gómez-González, Gabriela Hurtado-Alvarado and Javier Velázquez-Moctezuma Neuroscience Area, Dept. of Reproduction Biology, CBS, Universidad Autonoma Metropolitana, Unidad Iztapalapa, Mexico

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ABSTRACT The blood-brain barrier (BBB) is a highly regulated system that maintains brain allostasis. BBB achieves its main function by transporting from blood to brain glucose, amino acids and other molecules needed for proper neural physiology, while extruding from the brain molecules derived from neural and glial metabolism that may have neurotoxic properties. BBB function is tightly regulated by local synaptic and glial activity; both nerve and glial cells release molecules that modify hemodynamic and permeability parameters at the BBB. Under normal physiological conditions, products released by neural activity, such as some neurotransmitter/neuromodulator molecules and byproducts of neural metabolism, exert vasodilator effects and induce changes in BBB permeability. Active neurons have been shown to release several molecules that quickly increase cerebral blood flow locally, with a concomitant rise in BBB permeability to glucose and other solutes. In order to achieve prolonged activity-related effects on cerebral blood-flow and vessel permeability astrocytes are needed. Astrocyte calcium waves induce the release of vasoactive molecules that exert long-term hemodynamic and permeability changes in local vessels. Under altered physiological conditions, BBB function is also modified in a brain region specific manner. The mediators of the changes are almost the same as those of the normal physiological state, but their effects are intensenly and prolonged. Stress, a state of disrupted allostasis, has been shown to increase BBB permeability to blood-borne potentially neurotoxic molecules through increased vesicle-mediated transport at adulthood and during early-life; the effects are observed in some cortical regions, the brain stem, cerebellum, hippocampus, basal ganglia, and cervical spinal cord. Both adult

The Blood-Brain Barrier: New Research : New Research, edited by Pedro A. Montenegro, and Stefanee M. Juárez, Nova Science Publishers,

2

B. Gómez-González, G. Hurtado-Alvarado and J. Velázquez-Moctezuma and early-life stress exerted its effects upon BBB through increased release of serotonin, corticotrophin-releasing hormone, and pro-inflammatory cytokines. Sleep restriction is another altered physiological state that is accompanied by increased BBB permeability to blood-borne potentially toxic molecules. In a rat model, sleep restriction to 4-hours per day increased BBB permeability to an albumin-bound dye in almost the whole brain; the mechanism of the increased vessel permeability may be sleep deprivation-related hyperthermia, increased pro-inflammatory cytokine production and maybe some byproduct of altered neural and glial function after chronic sleep restriction. In conclusion, BBB function is tightly regulated inclusive during altered physiological states, some brain regions are affected during stress exposure or sleep restriction but others are spared and have normal brain vessel permeability. More research is needed to clarify regional differences in BBB properties and to elucidate the temporal organization of the involved molecules.

INTRODUCTION

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The blood-brain barrier is the key structure that maintains central nervous system allostasis.

Figure 1. Blood-brain barrier structure during normal physiological conditions. The blood-brain barrier provides a stable environment for neuronal function. Pericytes are small cells, analogous to perivascular smooth muscle cells (SMC), located at the outside of the microvessel, they are separated from the parenchyma by the basement membrane (BM). Foot processes from astrocytes form a complex network surrounding the capillaries. Microglia (MG) is the first line of defense in central nervous system. Brain endothelial cells synthesize proteoglycans and glycosaminoglycans, which form the glycocalyx (Gx). Besides being blood-brain barrier components, these cells together with neurons constitute the neurovascular unit and release vasoactive agents that exert both vasoconstrictor and vasodilatation effects.

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Local and Temporal Regulation …

3

Blood-brain barrier exerts that function by: (1) tightly regulating blood-to-brain transport of diverse molecules used as nutrients and metabolic substrates by neurons and glia, (2) hindering the brain parenchyma of the potentially neurotoxic blood-circulating molecules, (3) degrading or extruding brain-borne toxic molecules, and (4) allowing and promoting bidirectional signaling between the brain and the periphery (Fishman, 1990; Spector, 2010). Blood-brain barrier localizes in nearly 99% of brain microvessels (Patcher et al., 2003), therefore it constitutes a 150-200 cm2 g-1 surface area for molecular exchange (Nag and Beagley, 2005). The rest 1% of the brain microvessels are located in regions specialized in the free exchange of macromolecules from blood to brain and vice versa, and are globally called circunventricular organs (which include the choroid plexus, the pineal gland, the posterior lobe of the pituitary, the median eminence, the area postrema, the subfornical, subcommisural, and paraventricular organs and the vascular organ of the lamina terminallis (Patcher et al., 2003; Joly et al., 2007). In those regions brain capillaries do not express the blood-brain barrier phenotype (Norsted et al., 2008) and also present fenestrae that allow bidirectional transport of molecules (Peters et al., 1991; Ueno et al., 2000). The blood-brain barrier is a complex structure formed by different cell types that include endothelial cells, astrocytes, pericytes, and microglia; also, it is composed of non-cellular components, such as the glycocalyx and the basal lamina (Figure 1) (Risau, 1991; Ballabh et al., 2004; Ueno, 2007). The main cell type that confers the physical and chemical barrier function to the blood-brain barrier is the endothelial cell; however, other cell types are needed to develop and maintain the barrier function of the blood-brain barrier (Risau, 1991; Abbot et al., 2006).

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BLOOD-BRAIN BARRIER STRUCTURE DURING NORMAL PHYSIOLOGICAL CONDITIONS Endothelial Cells Brain microvessels are formed by a monolayer of endothelial cells (Peters et al., 1991; Ueno, 2007) constituting a wall of about 500nm thick (Zlokovic, 2008; Abbot et al., 2010). Brain parenchyma capillaries are derived from the progressive ramification of the Willis polygon extracerebral arteries into radial penetrating arterioles (10-60m in diameter), that subsequently divide into less than 10m diameter vessels (Edvinsson and Mackenzie, 2002). Brain capillaries are localized in close contact to neurons; in the rat hippocampus it is known that the average distance between a microvessel and a neuron is about 8-23m (Lovick et al., 1999) and it is estimated that each capillary perfuse a surrounding tissue volume of 15-50m diameter (Hamilton et al., 2010). Brain endothelial cells express many key features that confer them the physical and chemical barrier properties of the blood-brain barrier; brain capillaries are continuous (have no fenestrae) and establish an intercellular junctional complex (composed of tight and adherens junctions) that impedes interendothelial diffusion of hydrophilic molecules. Tight junctions are located at the apical pole of the endothelial cell; meanwhile, adherens junctions define the basolateral pole of the endothelial cell (Abbot et al., 2006; Zlokovic, 2008). Other hallmarks of the brain endothelial cell include low vesiclemediated transport (Peters et al., 1991; Abbot et al., 2010), the presence of specialized carrier

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systems for the transport of glucose, amino acids and other solutes from blood to brain and numerous extrusion systems that transport molecules from brain to blood (Zlokovic, 2008; Lourenço et al., 2010). Capillaries are distributed heterogeneously in the brain parenchyma, i.e. a higher density of capillaries is observed in gray matter as compared to white matter (Peters et al., 1991). Furthermore, inside the same micro-region, capillary density varies as a function of synaptic activity. In the primary sensory cortices capillaries are denser in the layer IV (where thalamic afferents are located) as compared to the rest of the cortical layers (Patel, 1983; Woolsey et al., 1996; Zheng et al., 1991). Capillary density is also higher inside barrels of the primary somatosensory cortex as compared to interbarrel cortical layers (Patel, 1983; Woolsey et al., 1996) and inside olfactory bulb glomeruli as compared to interglomerular spaces in the rat (Borowsky and Collins, 1989). The activity-dependent capillary density emerge since early postnatal life in mammals; in the rat cerebral cortex, microvasculature develops earlier in the most primitive regions and also those regions show a higher capillary density as compared to late-generated regions at a given time point (Rowan and Maxwell, 1981). Moreover, early-life modification of the afferents to a specific cortical region shapes microvascular pattern and blood-brain barrier phenotype. In the rat, exposure to visually enriched environments increased both vascular density and the expression of blood-brain barrier markers in the primary visual cortex as compared to standard reared rat pups; meanwhile rat pups reared in total darkness showed delayed microvascular development accompanied by decreased capillary density and diminished expression of blood-brain barrier markers in the same cortical region (Argandoña and Lafuente, 1996; Argandoña et al., 2005). The influence of local synaptic activity in the microvasculature occurs not only at the structural level (accomplished by increased or decreased capillary density in specific brain regions) but also at the physiological level. In the mammalian brain it has been shown that increased neuronal activity induces regional increases in cerebral blood flow (CBF), glucose and oxygen consumption temporally restricted to the duration of the increased neuronal activity (Filosa and Blanco, 2007; Cauli and Hamel, 2010; Figley and Stroman, 2011); that synaptic-related CBF change is named functional hyperaemia or neurovascular coupling (Mosso, 1880; Roy and Sherrington, 1890; Filosa and Blanco, 2007; Cauli and Hamel, 2010). Moreover, hemodynamic changes are also observed before actual increases in local activity; in the rat the search for potentially stimuli sources (by locomotion toward an object and whisker behavior) lead to increases in CBF at the primary somatosensory cortex (Ferezou et al., 2006). Neurovascular coupling occurs at the level of peri-capillary arterioles and is followed by neurometabolic coupling between neurons and glia, and by neurobarrier coupling at the local capillaries (Leybaert, 2005; Magistretti, 2006; Leybaert et al., 2007). The increased local blood flow in synaptic active regions depends on vasodilatation as well as on the passage from low-perfused to high-perfused capillaries (termed functional recruitment) (Paulson, 2002). Neurometabolic coupling implies that astrocytes deliver to active neurons adequate amounts of energy substrates; that function is accomplished by the astrocyte-mediated transport of glucose from blood to neurons and also by the delivery to neurons of lactate obtained from astrocyte glycolysis (Magistretti and Pellerin, 1999; Magistretti et al., 1999; Magistretti, 2006). While neurobarrier coupling implies that at the blood brain barrier significant changes in the rate of transport of glucose and other metabolic substrates occur in response to increased neuronal activity (Leybaert, 2005; Leybaert et al., 2007). All those three

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processes (neurovascular, neurometabolic and neurobarrier coupling) ensure that adequate supply of glucose, oxygen and other molecules is carried on to highly active neural regions (Leybaert, 2005; Leybaert et al., 2007; Cauli and Hamel, 2010) and as such, they require the strictly regulated temporal and local participation of the other blood-brain barrier cellular components (Figure 1).

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Pericytes As part of the lineage of perivascular smooth muscle cells, pericytes are localized in the external wall of small vessels, including arterioles, capillaries and venules, in the periphery and in the central nervous system (Figure 1). Pericytes cover around 20 to 30% of brain capillary external surface and are outnumbered by endothelial cells in a ratio 5:1 (Balanov and Dore-Duffy, 1998; Nag, 2003; Lai and Kuo, 2005; Kim et al., 2006). Two pericyte types are distinguished at brain capillaries, granular and agranular pericytes; ultrastructurally, granular pericytes show a higher density of lysosomes and endosomes than agranular pericytes (Nag, 2003). Because of their “granular” appearance and of their establishment of gap junctions with endothelial cells, pericytes are thought to participate in the phagocytosis of blood-components that may have passed across the endothelial cell membrane (Sims, 1986; Thomas, 1999). In addition to gap junctions, pericytes form tight junctions, adherens junctions and adhesion plaques with brain endothelial cells (Allt and Lawrenson, 2001; Nag, 2003; Zlokovic, 2008); those junctional complexes contribute to maintain the low permeability properties of brain endothelial cells by providing structural support and maintaining endothelial junction integrity (Lai and Kuo, 2005). Pericytes also release vasoactive factors, such as the fibroblast growth factor (FGF), the vascular endothelial growth factor (VEGF), angiopoietin-1, and transforming growth factor  (TGF) that promote endothelial cell proliferation, differentiation and the expression of the efflux systems (i.e. the P-glycoprotein) (Sims, 1986; Shepro and Morel, 1993; Dohgu et al., 2005; Shimizu et al., 2008). Pericytes are embedded inside the basal lamina, they send out gross cytoplasmic processes (300-800nm thick) around brain vessels (Figure 1) (Sims, 1986; Shepro and Morel, 1993; Balanov and Dore-Duffy, 1998). Pericyte primary cytoplasmic extensions originate from cell poles and subsequently ramify into secondary and tertiary extensions that surround brain microvessels. Both their localization around brain microvessels and their high expression of the -smooth muscle actin (SMA) (Shepro and Morel, 1993; Toribatake et al., 1997) situate pericytes as mediators of the rapid changes observed in CBF associated to local synaptic activity (Hamilton et al., 2010). Pericytes have been shown to induce vasoconstriction and vasodilatation at arteriolar and capillary vessels (Pappiat et al., 2006). Activity-dependent vascular changes have been described to occur at different levels of brain vasculature, since large extracerebral arteries until small capillaries. Vasodynamic properties of both large and small brain vessels are tightly regulated by numerous chemical signals coming extrinsically from periphery or intrinsically from brain; those changes occur throughout contraction or relaxation of perivascular smooth muscle cells in large extracerebral vessels and of pericytes in small parenchymal vessels (Bleys and Cowen, 2001; Hamilton et al., 2010). Periphery signals regulating vasodynamic properties of large vessels are of two types, blood-circulating signals that contribute to vasodilatation or vasoconstriction

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in the Willis circle arteries as a function of global physiology of the whole organism (Sharma et al., 1996; Huber et al., 2001a; Esposito et al., 2001, 2002); and sympathetic and parasympathetic innervation that locally produce vasoconstriction or vasodilatation, respectively (Edvinsson et al., 1987; Bleys and Cowen, 2001). Sympathetic nerves innervating large extracerebral vessels have been shown to release noradrenaline, Y neuropeptide and serotonin (Edvinsson et al., 1987); meanwhile, parasympathetic nerves release nitric oxide (NO) as a main effector and acetylcholine and vasoactive intestinal peptide (VIP) as modulators of vasodilatation (Lee, 2000). Intrinsic innervation of brain arteries and parenchymal arterioles includes nerve terminals arising from the locus coeruleus, the dorsal raphe nucleus, the tractus solitarius nucleus and the basal forebrain cholinergic nuclei (Kalaria et al., 1989; Reis and Iadecola, 1986; Sato and Sato, 1992). At the capillary level, it has been shown that in close contact with pericytes there are nerve terminals containing dopamine, VIP, acetylcholine, NO, and -amino butyric acid (GABA) (Arneric et al., 1988; Gragera et al., 1993; Roufail et al., 1995; Benagiano et al., 1996; Krimer et al., 1998). Neurotransmitters such as histamine, noradrenaline, and serotonin induce pericyte constriction in vitro (Kelley et al., 1988; Markhotina et al., 2007); meanwhile, VIP, NO, adenosine, and the drop in extracellular pH induce pericyte dilatation also in in vitro experiments (Haefliger et al., 1994; Matsugi et al., 1997; Chen and Andreson, 1997; Markhotina et al., 2007). In addition to neural-derived signals, pericytes sense the drop in oxygen concentration and the rise in lactate and adenosine at the interstitial fluid (both byproducts of neuronal and astroglial metabolism); those extracellular signals induce pericyte dilatation with a concomitant local increase in CBF and nutrient influx (Hamilton et al., 2010; Carmignoto and Gómez-Gonzalo, 2010). Like in the vascular smooth muscle cells, molecules generating constriction of the pericytes induce that by depolarizing the cell throughout a cytoplasmic increase in calcium concentration (Sakagami et al., 1999). Molecules inducing pericyte constriction lead to voltage-dependent calcium channel opening and, secondarily to the opening of chloride channels, which due to the high cytoplasmic concentration of chloride ions lead to a higher pericyte depolarization and a huge vasoconstriction (Kawamura et al., 2002). On the other hand, pericyte dilatation is accomplished by cell hyperpolarization; that membrane potential change is produced by a decrease in intracellular calcium concentration, and also by two mechanisms of potassium efflux, one dependent on voltage-gated potassium channel opening and other related to the extracellular increase in potassium concentration as byproduct of astrocyte calcium waves (Filosa et al., 2006; Hamilton et al., 2010). Activitydependent vasodynamic changes at the capillary level situate pericytes as protagonists of neurovascular coupling (Hamilton et al., 2010); in the rat olfactory bulb it has been shown that activity-dependent CBF increases (by odor exposure) occur only at the level of capillaries irrigating the stimulated glomerulus instead of occurring at the upstream arterioles irrigating also neighborhood glomeruli (Chaigneau et al., 2003). By promoting such dramatic changes at the endothelial surface area, pericytes may also participate in neurobarrier coupling; however until now that role has been full-filled by astrocytes.

Astrocytes Astrocytes are glial cells derived from the neural ectoderm, which distribute heterogeneously in the mammalian brain. There are many astrocyte types based on their

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distinct morphology and properties (Matyash and Kettenmann, 2011); in mammalian brain it is considered that astroglia diversity rises up to 10 types named tanycytes, radial cells, Bergmann glia, protoplasmic astrocytes, fibrous astrocytes, velate glia, marginal glia, perivascular glia, ependymal glia, and interlaminal astrocytes (Colombo and Reisin, 2004; Emsley and Macklis, 2006). The ratio of astroglia to neuron varies from one to another species, in rodents is around 0.3 while in humans is up to 1.65 (Sherwood et al., 2006). As one cellular constituent of the blood-brain barrier, astrocytes participate actively in the maintenance of brain allostasis; astrocytes regulate ionic equilibrium and pH by sequestering potassium from the brain interstitial fluid. Astrocytes take part in the synthesis, release, and removal of glutamate and GABA from the synapses (Ransom et al., 2003). Astrocytes also release many neuro- and vasoactive molecules, such as taurin, aspartate, eicosanoids, neuropeptides (Martin, 1992), NO (Li et al., 2003), cyclooxygenase and epoxygenase derivatives (Amruthesh et al., 1992), ATP (Queiroz et al., 1999), cholesterol (Mauch et al., 2001), glial-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), angiopoietin-1 and TGF in order to accomplish neurovascular coupling and maintain the blood-brain barrier phenotype in endothelial cells (Abbot, 2002; Abbot et al., 2006; Weiss et al., 2009). Typically gray matter astrocytes possess between 5-8 primary ramifications, which divide into secondary and tertiary processes; astrocyte ramifications form microdomains of 300400m diameter that enclose between 20 000 to 120 000 synapses (Nedergaard et al., 2003; Oberheim et al., 2009). Astrocyte microdomains patches the neuropil into small independent functional unities; in response to increased neuronal activity, astrocytes present intracellular calcium waves that signal retrogradely to neurons by inducing astrocyte release of gliotransmitters into the tripartite synapse (Nedergaard, 1994; Fellin, 2009; Oberheim et al., 2009; Carmignoto and Gómez-Gonzalo, 2010). Astrocytes also extend cytoplasmic processes that enwrap brain capillaries; those astrocyte perivascular endfeet cover around 85-99% of external capillary surface (Peters et al., 1991; Risau, 1991; Agulhon et al., 2008). Therefore astrocytes are situated not only as the main mediators of local neural excitability and synaptic transmission, but also as protagonists of neurovascular, neurometabolic, and neurobarrier coupling (Figure 1). As a component of the tripartite synapse, astrocytes sense local neuronal activity by the activation of their membrane receptors to neurotransmitters such as glutamate, GABA, Y neuropeptide, somatostatin, VIP, and ATP (Carmignoto and Gómez-Gonzalo, 2010). Astrocytes also sense the interstitial fluid concentration of oxygen, which varies as a function of local neuronal activity. Early after neural stimulation (300-500 milliseconds delay) there is a local decrease in oxygen concentration followed by a rapid rise due to vasodilatation associated to neuronal activity (Yamamoto and Kato, 2002; Carmignoto and Gómez-Gonzalo, 2010). In response to both receptor activation and the transport of glutamate and GABA into the astrocyte cytoplasm, calcium is released from intracellular stores and calcium waves propagate to the perivascular endfeet (Zonta et al., 2003a, 2003b). The intracellular accumulation of calcium induces astrocyte release of arachidonic acid derivatives (Amruthesh et al., 1992; Zonta et al., 2003a), potassium (Filosa et al., 2006), NO (Metea and Newman, 2006), and other modulator molecules from the perivascular endfeet (Gordon et al., 2008). Under low oxygen concentration activity-related astrocyte calcium waves induce the release of lactate and prostaglandin E2 (PGE2). Lactate conjointly with adenosine (extracellularly accumulated by the decreased oxygen concentration) potentialize PGE2 mediated

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vasodilatation; lactate inhibits prostaglandin transporters and adenosine down-regulates Ltype calcium channels activity at the perivascular smooth muscle cells and pericytes leading to vasodilatation (Gordon et al., 2008). Free interstitial potassium released by astrocytes also promotes pericyte and perivascular smooth muscle cell dilatation (Filosa et al., 2006). By the contrary, under high oxygen concentration, astrocyte calcium waves induce vasoconstriction due to the conversion of arachidonic acid into 20-hydroxyeicosatetraenoic acid (20-HETE) stimulated by astrocyte NO. The 20-HETE has been shown to exert strong vasoconstrictor effects (Metea and Newman, 2006; Gordon et al., 2008). Although astrocytes play a major role in generating functional hyperaemia, findings obtained in time course studies indicate that activity-dependent neurovascular coupling occurs throughout two mechanisms, one dependent on astrocyte activity and the other dependent on neuronal release of vasoactive molecules, such as NO (Cauli and Hamel, 2010). For instance, in rat somatosensory cortex, calcium waves preceded CBF changes after mechanical limb stimulation (Winship et al., 2007); hemodynamic changes were observed within 1 second after limb stimulation and the latency to the induction of astrocyte-calcium waves was only 0.5 seconds (Winship et al., 2007). However, the generally observed time course of neurovascular coupling implies a rapid-onset change in local blood flow (