Blood-Brain Barriers: From Ontogeny to Artificial Interfaces

Blood-Brain Barriers Edited by Rolf Dermietzel, David C.Spray, Maiken Nedergaard Blood-Brain Interfaces: From Ontogeny...

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Blood-Brain Barriers Edited by Rolf Dermietzel, David C.Spray, Maiken Nedergaard

Blood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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Blood-Brain Barriers From Ontogeny to Artificial Interfaces Volume 1

Edited by Rolf Dermietzel, David C.Spray, Maiken Nedergaard

Editors: Prof. Dr. Rolf Dermietzel Department of Neuroanatomy and Molecular Brain Research Ruhr University Bochum Universitätsstrasse 150 44780 Bochum Germany Prof. Dr. David Spray Department of Neuroscience Albert Einstein College of Medicine 1410 Pelham Parkway S Bronx, NY 10464 USA Prof. Dr. Maiken Nedergaard School of Medicine and Dentistry University of Rochester 601 Elmwood Avenue Rochester, NY 14642 USA

n All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data: A catalogue record for this book is available from the British Library Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the internet at http://dnb.ddb.de © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany All rights reserved (including those of translation in other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Typesetting K+V Fotosatz GmbH, Beerfelden Printing betz-druck GmbH, Darmstadt Binding Litges & Dopf Buchbinderei GmbH, Heppenheim Printed in the Federal Republic of Germany Printed on acid-free paper ISBN-13: 978-3-527-31088-3 ISBN-10: 3-527-31088-6

V

This handbook is dedicated to Eva

VII

Contents Preface

XXIII

List of Contributors

XXV

VOLUME 1 Introduction The Blood-Brain Barrier: An Integrated Concept 1 Rolf Dermietzel, David C. Spray, and Maiken Nedergaard Part I

Ontogeny of the Blood-Brain Barrier 9

1

Development of the Blood-Brain Interface Britta Engelhardt

1.1 1.2 1.3 1.4 1.5 1.5.1 1.5.2 1.5.3 1.5.4 1.6 1.7

Introduction 11 Pioneering Research on the Blood-Brain Barrier 11 The Mature Blood-Brain Interface 13 Development of the CNS Vasculature 17 Differentiation of the Blood-Brain Barrier 20 Permeability 20 Transport Systems and Markers 21 Extracellular Matrix 23 Putative Inductive Mechanisms 23 Maintenance of the Blood-Brain Barrier 27 Outlook 28 References 29


11

VIII

Contents

2

Brain Angiogenesis and Barriergenesis 41 Jeong Ae Park, Yoon Kyung Choi, Sae-Won Kim, and Kyu-Won Kim

2.1 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.3 2.3.1 2.3.2

Introduction 41 Brain Angiogenesis 42 Hypoxia-Regulated HIF-1 in the Development of the Brain 42 Hypoxia-Inducible Factor 43 Hypoxia-Induced VEGF 46 Other Neuroglia-Derived Angiogenic Factors 47 Oxygenation in the Brain: Brain Barriergenesis 49 Cellular and Molecular Responses Following Brain Oxygenation Role of src-Suppressed C Kinase Substrate in the Induction of Barriergenesis 50 Barriergenic Factors in Perivascular Astrocytes and Pericytes Following Brain Oxygenation 51 Perspectives 53 References 55

2.3.3 2.4

49

3

Microvascular Influences on Progenitor Cell Mobilization and Fate in the Adult Brain 61 Christina Lilliehook and Steven A. Goldman

3.1 3.2 3.3 3.4

Introduction 61 Angiogenic Foci Persist in the Adult Brain 61 Neurotrophic Cytokines Can Be of Vascular Origin 62 Angiogenesis and Neurogenesis are Linked in the Adult Avian Brain 63 Angiogenesis-Neurogenesis Interactions in the Adult Mammalian Brain 65 Purinergic Signaling to Neural Progenitors Cells: the Gliovascular Unit as a Functional Entity 66 Nitric Oxide is a Local Modulator of Progenitor Cell Mobilization 67 Parenchymal Neural Progenitor Cells May Reside Among Microvascular Pericytes 68 The Role of the Vasculature in Post-Ischemic Mobilization of Progenitor Cells 69 References 70

3.5 3.6 3.7 3.8 3.9

Contents

75

Part II

The Cells of the Blood-Brain Interface

4

The Endothelial Frontier 77 Hartwig Wolburg

4.1 4.2 4.3 4.3.1 4.3.1.1 4.3.1.2 4.3.2 4.3.3 4.4 4.5

Introduction 77 The Brain Capillary Endothelial Cell 78 Endothelial Structures Regulating Transendothelial Permeability 83 Tight Junctions 83 Morphology of Tight Junctions 83 Molecular Biology of Tight Junctions 84 Caveolae 89 Transporters in the Blood-Brain Barrier Endothelium 92 Brief Consideration of the Neuroglio-Vascular Complex 94 Conclusions 98 References 99

5

Pericytes and Their Contribution to the Blood-Brain Barrier 109 Markus Ramsauer

5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.8.1 5.8.2 5.8.3 5.9

Introduction 109 Pericyte Structure and Positioning 110 Pericyte Markers 113 Pericytes in Culture 113 Contractility and Regulation of Blood Flow 115 Macrophage Function 116 Regulation of Homeostasis and Integrity 118 Angiogenesis and Stability 120 PDGF-B and Pericyte Recruitment 120 TGF-b1 and Differentiation 121 Ang-1 and Maturation 122 Conclusion 123 References 123

6

Brain Macrophages: Enigmas and Conundrums 129 Frederic Mercier, Sebastien Mambie, and Glenn I. Hatton

6.1 6.2 6.2.1 6.2.1.1 6.2.1.2 6.2.1.3 6.2.1.4 6.2.2 6.2.3

Introduction 129 Different Types and Locations of Brain Macrophages 130 Macrophage Structure and Ultrastructure 133 Perivascular Macrophages 133 Meningeal Macrophages 138 Dendritic Cells 138 Ventricular Macrophages 139 Immunotyping by Cell Surface Antigens 139 Macrophages Contact Basal Laminae 140

IX

X

Contents

6.2.4 6.3 6.4 6.5 6.5.1 6.5.1.1 6.5.1.2 6.5.1.3 6.5.1.4 6.5.1.5 6.5.2 6.5.2.1 6.5.2.2 6.5.2.3 6.5.2.4 6.6

Network of Macrophages Through the Brain 141 Migration of Brain Macrophages 142 Fast Renewal of Brain Macrophages 142 Functions 143 Known Functions of Brain Macrophages 143 Phagocytosis 144 Immune Function 144 Production of Growth Factors, Cytokines, and Chemokines 144 Production and Degradation of the Extracellular Matrix 145 Repair After Injury 145 Potential Functions of Brain Macrophages 146 Interactions with Meningeal/Vascular Cells, Neurons, and Astrocytes 146 Do Macrophages Govern the Neural Stem Cell Niche in Adulthood? 147 Role of Macrophages in CNS Angiogenesis 151 Role of Macrophages in CNS Plasticity 152 Conclusion: Macrophages as Architects of the CNS Throughout Adulthood 153 References 154

7

The Microglial Component 167 Ingo Bechmann, Angelika Rappert, Josef Priller, and Robert Nitsch

7.1 7.1.1 7.1.2 7.1.3 7.2

Microglia: Intrinsic Immune Sensor Cells of the CNS 167 Development 167 Microglial Activation 168 Antigen Presentation/Cytotoxicity 169 Terminology: Subtypes and Their Location in Regard to Brain Vessels 170 Perivascular Macrophages 170 Juxtavascular and Other Microglia 170 Turnover of Brain Mononuclear Cells by Precursor Recruitment Across the BBB 173 Perivascular Cells 173 Microglia 174 Turnover Used by “Trojan Horses” 174 Microglial Impact on BBB Function 175 Concept of the BBB 175 Chemokines – an Overview 176 GAG/Duffy 176 Chemokine Expression in the CNS 177 CCL2 and CCR2 177 CCL3 and CCL5 178 CXCR3 and CXCL10 178

7.2.1 7.2.2 7.3 7.3.1 7.3.2 7.3.3 7.4 7.4.1 7.4.2 7.4.3 7.4.4 7.4.5 7.4.6 7.4.7

Contents

7.4.8 7.4.9 7.5

Microglia-Endothelial Cell Dialogue 179 Microglial Effects on Tight Junctions 179 Concluding Remarks 180 References 181

8

The Bipolar Astrocyte: Polarized Features of Astrocytic Glia Underlying Physiology, with Particular Reference to the Blood-Brain Barrier 189 N. Joan Abbott

8.1 8.2 8.3 8.4 8.5 8.6 8.7

Introduction 189 Formation of the Neural Tube 189 Origin of Neurons and Glia 190 Morphology of Glial Polarity in Adult CNS 193 Astrocyte Spacing and Boundary Layers 195 Origin and Molecular Basis of Cell Polarity 196 Functional Polarity of Astrocytes and Other Ependymoglial Derivatives 197 Secretory Functions of Astrocytes 199 Induction of BBB Properties in Brain Endothelium 199 Astrocyte-Endothelial Signaling 202 Conclusion 202 References 203

8.8 8.9 8.10 8.11

9

Responsive Astrocytic Endfeet: the Role of AQP4 in BBB Development and Functioning 209 Grazia P. Nicchia, Beatrice Nico, Laura M. A. Camassa, Maria G. Mola, Domenico Ribatti, David C. Spray, Alejandra Bosco, Maria Svelto, and Antonio Frigeri

9.1 9.2 9.3 9.4 9.5 9.5.1 9.5.2 9.5.2.1 9.5.2.2 9.5.2.3 9.5.2.4 9.6

Introduction 209 Astrocyte Endfeet and BBB Maintenance 210 Astrocyte Endfeet and BBB Development 212 Astrocyte Endfeet and BBB Damage 215 The Role of Aquaporins in BBB Maintenance and Brain Edema AQP Expression and Functional Roles 216 The Role of AQP4 in Brain Edema 220 DMD Animal Models 220 The a-Syntrophin Null Mice 223 The Effect of Lipopolysaccharide on AQP4 Expression 224 AQP4 in Astrocytomas 224 AQP4 Expression in Astrocyte-Endothelial Cocultures 225 References 230

216

XI

XII

Contents

237

Part III

Hormonal and Enzymatic Control of Brain Vessels

10

The Role of Fibroblast Growth Factor 2 in the Establishment and Maintenance of the Blood-Brain Barrier 239 Bernhard Reuss

10.1 10.2 10.2.1

Introduction 239 Role of FGF-2 in the Regulation of BBB Formation 239 Expression of FGF-2 in Astrocytes and Endothelial Cells of the Rodent Brain 239 Induction of BBB Properties in Endothelial Cells by Soluble Factors 240 Indirect Astrocyte Mediated Effects Seem to Play a Role in FGF-2-Dependent Changes in Endothelial Cell Differentiation 241 Involvement of FGF-2 in the Regulation of BBB Properties in the Pathologically Altered Brain 242 Future Perspectives 243 References 244

10.2.2 10.2.3

10.2.4 10.3

11

Cytokines Interact with the Blood-Brain Barrier 247 Weihong Pan, Shulin Xiang, Hong Tu, and Abba J. Kastin

11.1 11.2 11.2.1 11.2.2 11.2.3 11.3 11.3.1

Introduction 247 Identification of the Phenomena 248 Cytokines That Cross the BBB by Specific Transport Systems 248 Cytokines That Permeate the BBB by Simple Diffusion 249 Cytokines That Have Known Effects on Endothelial Cells 250 Mechanisms of Cytokine Interactions with the BBB 253 Endocytosis of Cytokines by the Apical Surface of Endothelial Cells 253 Intracellular Trafficking Pathways 253 Signal Transduction in Endothelial Cells 254 Involvement of Other Cells Comprising the BBB 254 Regulation of the Interactions of Cytokines with the BBB 254 Stroke and Other Vasculopathy 255 Neurodegenerative Disorders 256 Summary 257 References 258

11.3.2 11.3.3 11.3.4 11.4 11.5 11.6 11.7

Contents

12

Insulin and the Blood-Brain Barrier 265 William A. Banks and Wee Shiong Lim

12.1 12.1.1 12.1.2

Introduction 265 Early Studies 265 Debates Related to the Question of Permeability of the BBB to Insulin 269 Does Insulin Cross the BBB? The Middle Years 270 Insulin, the BBB, and Pathophysiology: The Past Decade 272 Pathophysiology of Insulin Transport 274 Insulin, Obesity, and Diabetes 274 Insulin Resistance and Inflammatory States 275 Insulin and Alzheimer’s Disease 277 References 280

12.1.3 12.1.4 12.2 12.2.1 12.2.2 12.2.3

13

Glucocorticoid Hormones and Estrogens: Their Interaction with the Endothelial Cells of the Blood-Brain Barrier 287 Jean-Bernard Dietrich

13.1 13.2 13.2.1 13.2.2 13.2.3

Introduction 287 Glucocorticoids and the Endothelial Cells of the BBB 288 Mechanisms of Action of Glucocorticoids 288 Glucocorticoids and Inflammation 289 Regulation of Adhesion Molecules Expression by GC in Endothelial Cells 290 Effects of GC on Leukocyte-Endothelial Cell Interactions 293 Glucocorticoids, Cerebral Endothelium and Multiple Sclerosis 294 Estrogens and the Endothelial Cells of the BBB 297 Mechanisms of Action of Estrogens 297 Endothelial Cells as Targets of Estrogens 298 Adhesion Molecules are Regulated by Estrogens in Endothelial Cells 298 Estrogens and Experimental Autoimmune Encephalomyelitis 299 Conclusions and Perspectives 301 References 302

13.2.4 13.2.5 13.3 13.3.1 13.3.2 13.3.3 13.3.4 13.4

313

14

Metalloproteinases and the Brain Microvasculature Dorothee Krause and Christina Lohmann

14.1 14.2 14.3 14.4 14.4.1 14.4.2 14.5

Introduction 313 Metalloproteinases in Brain Microvessels: Types and Functions 314 Cerebral Endothelial Cells and Metalloproteinases 318 Perivascular Cells and Metalloproteinases 321 Pericytes 321 Astrocytes and Microglia 322 Metalloproteinases and the Blood-Liquor Barrier 324

XIII

XIV

Contents

14.6 14.6.1 14.6.2 14.6.3 14.6.4 14.7

Metalloproteinases and Brain Diseases 325 Metalloproteinases and Cerebral Ischemia 326 Metalloproteinases and Brain Tumors 327 Metalloproteinases and Multiple Sclerosis 327 MMPs and Migraine 328 Conclusion 328 References 328

Part IV

Culturing the Blood-Brain Barrier 335

15

Modeling the Blood-Brain Barrier 337 Roméo Cecchelli, Caroline Coisne, Lucie Dehouck, Florence Miller, Marie-Pierre Dehouck, Valérie Buée-Scherrer, and Bénédicte Dehouck

15.1 15.1.1 15.1.2

Introduction 337 In Vitro BBB Model Interests 337 The BBB: Brain Capillary Endothelial Cells and Brain Parenchyma Cells 338 Culturing Brain Capillary Endothelial Cells 338 Brain Capillary Endothelial Cell Isolation 338 Brain Capillary Endothelial Cell Isolation 338 Endothelial Cell Culture From Capillaries 340 Primary Endothelial Cells and Subculture of Brain Capillary Endothelial Cells 341 Immortalization 342 Purity 344 Species 346 Coculture 347 Characteristics Required for a Useful In Vitro BBB Model 347 Confluent Monolayer 347 Tight Junctions and Paracellular Permeability 349 Transcellular Transport, Receptor Mediated Transport 350 Expression of Endothelial Adhesion Molecules/Vascular Inflammatory Markers 351 Conclusion 352 References 352

15.2 15.2.1 15.2.1.1 15.2.1.2 15.2.1.3 15.2.1.4 15.2.1.5 15.2.1.6 15.2.2 15.3 15.3.1 15.3.2 15.3.3 15.3.4 15.4

16

Induction of Blood-Brain Barrier Properties in Cultured Endothelial Cells 357 Alla Zozulya, Christian Weidenfeller, and Hans-Joachim Galla

16.1 16.2 16.3

Introduction 357 In Vitro BBB Models 359 Hydrocortisone Reinforces the Barrier Properties of Primary Cultured Cerebral Endothelial Cells 360

Contents

16.3.1 16.3.2 16.4 16.5 16.5.1 16.5.2 16.6 16.7

In Vitro Model Based on Pig Brain Capillary Endothelial Cells (PBCEC) 360 In Vitro Model Based on Mouse Brain Capillary Endothelial Cells (MBCEC) 361 The Involvement of Serum Effects 362 Hydrocortisone Improves the Culture Substrate by Suppressing the Expression of Matrix Metalloproteinases In Vitro 363 ECIS Analysis of Improved Endothelial Cell-Cell and Cell-Substrate Contacts in HC-Supplemented Medium 363 Low Degradation of ECM in HC-Supplemented Medium Leads to Improved Cell-Substrate Contacts of Cerebral Endothelial Cells 365 The Role of Endogenously Derived ECM for the BBB Properties of Cerebral Endothelial Cells In Vitro 368 Conclusions 370 References 371

17

Artificial Blood-Brain Barriers 375 Luca Cucullo, Emily Oby, Kerri Hallene, Barbara Aumayr, Ed Rapp, and Damir Janigro

17.1 17.2 17.3 17.4 17.4.1 17.4.1.1 17.4.1.2 17.4.1.3 17.4.2 17.4.3 17.5 17.6 17.6.1 17.6.2 17.7 17.8

Introduction: The Blood-Brain Barrier 375 Requirements for a Good BBB Model 378 Immobilized Artificial Membranes 379 Cell Culture-Based in vitro BBB Models 379 Cell Lines 380 Immortalized Rat Brain Endothelial Cells 380 Other Cells From Non-Cerebral Sources 380 Brain Capillary Endothelial Cell Cultures 381 Monoculture-Based in vitro BBB Models 382 Coculture-Based in vitro BBB Models 385 Shear Stress and Cell Differentiation 386 Flow-Based in vitro BBB Systems 387 Dynamic in vitro BBB: Standard Model 387 Dynamic in vitro BBB: New Model 390 A Look Into The Future: Automated Flow Based in vitro BBBs 392 Conclusion 392 References 394

18

In Silico Prediction Models for Blood-Brain Barrier Permeation Gerhard F. Ecker and Christian R. Noe

18.1 18.2 18.3 18.4 18.5

Introduction: The In Silico World 403 The Blood-Brain Barrier 404 Data Sets Available 405 Computational Models 410 Passive Diffusion 410

403

XV

XVI

Contents

18.5.1 18.5.2 18.6 18.7 18.8

Regression Models 410 Classification Systems 419 Field-Based Methods 421 Active Transport 423 Conclusions and Future Directions 426 References 426

VOLUME 2 Part V

Drug Delivery to the Brain 429

19

The Blood-Brain Barrier: Roles of the Multidrug Resistance Transporter P-Glycoprotein 431 Sandra Turcotte, Michel Demeule, Anthony Régina, Chantal Fournier, Julie Jodoin, Albert Moghrabi, and Richard Béliveau

19.1 19.2 19.2.1 19.2.2 19.2.3 19.3 19.3.1 19.3.2 19.3.2.1 19.3.2.2 19.3.3 19.3.4 19.4 19.4.1 19.4.2 19.5 19.5.1 19.5.2

Introduction 431 The Multidrug Transporter P-Glycoprotein 432 P-gp Isoforms 432 Structure 433 P-gp Substrates 435 Localization and Transport Activity of P-gp in the CNS 438 Normal Brain 438 Brain Diseases 440 Malignant Brain Tumors 440 Brain Metastases 441 Expression of Other ABC Transporters at the BBB 442 Subcellular Localization of P-gp 442 Polymorphisms of P-gp 444 MDR1 Polymorphisms at the BBB 444 MDR1 Polymorphism and Brain Pathologies 445 Role of P-gp at the BBB 446 Protection Against Xenobiotics 448 Secretion of Endogenous Brain Substrates and Endothelial Secretion 448 Caveolar Trafficking 449 Conclusions 450 References 451

19.5.3 19.6

20

Targeting of Neuropharmaceuticals by Chemical Delivery Systems 463 Nicholas Bodor and Peter Buchwald

20.1 20.2

Introduction 463 The Blood-Brain Barrier 464

Contents

20.2.1 20.2.2 20.3 20.3.1 20.3.2 20.4 20.5 20.5.1 20.5.2 20.5.3 20.5.4 20.5.5 20.5.6 20.6 20.6.1 20.6.2 20.6.3 20.6.4

Structural Aspects 464 Enzymatic and Transporter-Related Aspects 466 Brain-Targeted Drug Delivery 467 Lipophilicity and Its Role in CNS Entry 468 Quantifying Brain-Targeting: Site-Targeting Index and Targeting Enhancement Factors 470 Chemical Delivery Systems 472 Brain-Targeting CDSs 473 Design Principles 473 Zidovudine-CDS 475 Ganciclovir-CDS 476 Benzylpenicillin-CDS 478 Estradiol-CDS 479 Cyclodextrin Complexes 484 Molecular Packaging 484 Leu-Enkephalin Analogs 485 TRH Analogs 486 Kyotorphin Analogs 487 Brain-Targeted Redox Analogs 489 References 490

21

Drug Delivery to the Brain by Internalizing Receptors at the Blood-Brain Barrier 501 Pieter J. Gaillard, Corine C. Visser, and Albertus (Bert) G. de Boer

21.1 21.2 21.3 21.4 21.5 21.6 21.7 21.8 21.9

Introduction 501 Blood-Brain Barrier Transport Opportunities 502 Drug Delivery and Targeting Strategies to the Brain 504 Receptor-Mediated Drug Delivery to the Brain 506 Transferrin Receptor 506 Insulin Receptor 508 LRP1 and LRP2 Receptors 509 Diphtheria Toxin Receptor 511 Conclusions 512 References 514

Part VI

Vascular Perfusion 521

22

Blood-Brain Transfer and Metabolism of Oxygen Albert Gjedde

22.1 22.2 22.2.1 22.2.2 22.3

Introduction 523 Blood-Brain Transfer of Oxygen 525 Capillary Model of Oxygen Transfer 525 Compartment Model of Oxygen Transfer 528 Oxygen in Brain Tissue 529

523

XVII

XVIII

Contents

22.3.1 22.3.2 22.3.2.1 22.3.2.2 22.4 22.5 22.5.1 22.5.2 22.6 22.6.1 22.6.2 22.6.3

Cytochrome Oxidation 529 Mitochondrial Oxygen Tension 531 Distributed Model of Tissue and Mitochondrial Oxygen 531 Compartment Models of Tissue and Mitochondrial Oxygen 533 Flow-Metabolism Coupling of Oxygen 535 Limits to Oxygen Supply 538 Distributed Model of Insufficient Oxygen Delivery 538 Compartment Model of Insufficient Oxygen Delivery 541 Experimental Results 542 Brain Tissue and Mitochondrial Oxygen Tensions 542 Flow-Metabolism Coupling 543 Ischemic Limits of Oxygen Diffusibility 546 References 547

23

Functional Brain Imaging Gerald A. Dienel

23.1 23.1.1 23.1.2 23.1.3 23.2 23.2.1 23.2.2 23.2.3 23.2.4 23.2.5 23.2.6 23.2.7 23.3

Molecular Imaging of Biological Processes in Living Brain 551 Introduction 551 Molecular Imaging 551 Influence of Blood-Brain Interface on Functional Imaging 552 Overview of Brain Imaging Methodologies 554 Computed Tomography 556 Magnetic Resonance Imaging 556 Functional MRI 557 Radionuclide Imaging 560 Optical Imaging 562 Thermal and Optico-Acoustic Imaging 564 Summary 565 Imaging Biological Processes in Living Brain: Watching and Measuring Brain Work 565 Functional Activity, Brain Work, and Metabolic Imaging 565 Quantitative Measurement of Regional Blood Flow and Metabolism in Living Brain 567 Assays at the Blood-Brain Interface: Global Methods 567 Highly Diffusible Tracers to Measure CBF 568 Metabolizable Glucose Analogs to Measure Hexokinase Activity and CMRglc 569 Non-Metabolizable Analogs to Assay Transport and Tissue Concentration 572 Cellular Basis of Glucose Utilization 572 Acetate is an “Astrocyte Reporter Molecule” 573 Summary 573 Molecular Probes are Used for a Broad Spectrum of Imaging Assays in Living Brain 574

23.3.1 23.3.2 23.3.2.1 23.3.2.2 23.3.2.3 23.3.2.4 23.3.2.5 23.3.2.6 23.3.2.7 23.4

551

Contents

23.4.1 23.4.2 23.4.3 23.4.4

23.4.5 23.4.6

23.4.7 23.5 23.6 23.7 23.8

Potassium Uptake and CMRglc During Functional Activation 574 Multimodal Assays in Serial Sections of Brain 574 Imaging Human Brain Tumors 576 Functional Imaging Studies of Sensory and Cognitive Activity Reveal Disproportionate Increases in CBF and CMRglc Compared to CMRO2 During Activation 576 Imaging Brain Maturation and Aging, Neurotransmitter Systems, and Effects of Drugs of Abuse 580 Imaging Electrolyte Transport Across the Blood-Brain Interface and Shifts in Calcium Homeostasis Under Pathophysiological Conditions 584 Summary 587 Optical Imaging of Functional Activity by Means of Extrinsic and Intrinsic Fluorescent Compounds 587 Tracking Dynamic Movement of Cellular Processes and Cell Types 589 Evaluation of Exogenous Genes, Cells, and Therapeutic Efficacy 590 Summary and Perspectives 594 References 595 601

Part VII

Disease-Related Response

24

Inflammatory Response of the Blood-Brain Interface Pedro M. Faustmann and Claus G. Haase

24.1 24.2 24.2.1 24.2.2 24.2.3 24.3 24.3.1 24.3.2 24.3.3 24.4 24.5

Introduction 603 Diagnostic Features of Cerebrospinal Fluid 603 Cell Count and Cell Differention 604 Protein Level 605 Glucose Level 605 Acute Bacterial Meningitis 605 Bacteria and the Blood-Brain Barrier 606 Leukocyte Migration into the CNS 607 Cytokines 611 Inflammatory Response in Acute Trauma 612 Inflammatory Response in Alzheimer’s Disease 613 References 614

25

Stroke and the Blood-Brain Interface Marilyn J. Cipolla 619

25.1 25.2 25.2.1 25.3 25.3.1

Introduction 619 Brain Edema Formation During Stroke 620 Cytotoxic Versus Vasogenic Edema 620 Role of Astrocytes in Mediating Edema During Ischemia 621 Aquaporins and Cerebral Edema During Ischemia 624

603

619

XIX

XX

Contents

25.4 25.4.1 25.4.2 25.5 25.5.1 25.5.2 25.6 25.7 25.8 25.8.1 25.9

Cellular Regulation of Cerebrovascular Permeability 626 Role of Actin 626 Ischemia and Hypoxia Effects on EC Actin and Permeability 627 Reperfusion Injury 628 Nitric Oxide and Other Reactive Oxygen Species as Mediators of EC Permeability During Ischemia And Reperfusion 628 Thresholds of Injury 629 Transcellular Transport as a Mechanism of BBB Disruption During Ischemia 630 Mediators of EC Permeability During Ischemia 631 Hyperglycemic Stroke 633 Role of PKC Activity in Mediating Enhanced BBB Permeability During Hyperglycemic Stroke 633 Hemodynamic Changes During Ischemia and Reperfusion and its Role in Cerebral Edema 634 References 635

26

Diabetes and the Consequences for the Blood-Brain Barrier 649 Arshag D. Mooradian

26.1 26.2 26.3 26.3.1 26.4 26.4.1 26.4.2 26.4.3 26.5 26.6

Introduction 649 Histological Changes in the Cerebral Microvessels 650 Functional Changes in the Blood-Brain Barrier 650 Diabetes-Related Changes in the BBB Transport Function 651 Potential Mechanisms of Changes in the BBB 655 Hemodynamic Changes 655 Biophysical and Biochemical Changes 656 Changes in Neurotransmitter Activity in Cerebral Capillaries 657 Potential Clinical Consequences of Changes in the BBB 658 Conclusions 661 References 661

27

Human Parasitic Disease in the Context of the Blood-Brain Barrier – Effects, Interactions, and Transgressions 671 Mahalia S. Desruisseaux, Louis M. Weiss, Herbert B. Tanowitz, Adam Mott, and Danny A. Milner

27.1 27.2

Introduction (by D. Milner) 671 Malaria: The Plasmodium berghei Mouse Model and the Severe Falciparum Malaria in Man (by M.S. Desruisseaux and D. Milner) 683 Upregulation of Intercellular Adhesion Molecules 685 Secretion of TNF-a 686 Microglial Activation 687 Vascular Damage 688

27.2.1 27.2.2 27.2.3 27.2.4

Contents

27.3

27.3.1 27.3.2 27.3.3 27.3.3.1 27.3.4 27.4 27.5

28

28.1 28.2 28.3 28.4 28.5 28.6 28.7 28.8 28.9 28.10 28.11

28.11.1 28.12 28.13

Trypanosomiasis: African and American Parasites of Two Distinct Flavors (by H. Tanowitz, M. S. Desruisseaux, and A. Mott) 689 CNS Pathology in African Trypanosomiasis 690 Cytokines and Endothelial Cell Activation 690 Astrocytosis and Microglial Cell Activation 691 Nitric Oxide 691 American Trypanosomiasis (Chagas’ Disease) 692 Toxoplasmosis: Transgression, Quiescence, and Destructive Infections (by L. Weiss) 694 Conclusion 696 References 696 The Blood Retinal Interface: Similarities and Contrasts with the Blood-Brain Interface Tailoi Chan-Ling

701

Introduction 701 The Inner and Outer BRB 702 The Choroidal Vasculature 702 Characteristics of Intraretinal Blood Vessels 703 Ensheathment and Induction of the Inner BRB by Astrocytes and Müller Glia 703 BRB Properties of Newly Formed Vessels 706 Pericytes and the BRB 707 Membrane Proteins of Tight Junctions 710 Localization of Occludin and Claudin-1 to Tight Junctions of Retinal Vascular Endothelial Cells 710 Expression of Occludin by RPE Cells and Lack of Occludin Expression by Choroidal Vessels 711 Inherent Weakness of the BRB and Existence of Resident MHC Class II+ Cells Predisposes the Optic Nerve Head to Inflammatory Attack 712 Compromised BBB Where CNS Meets Peripheral Vascular Bed 713 Clinical and Experimental Determination of the Blood-Retinal Barrier 713 Conclusions 716 References 717 Subject Index

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Preface Writing on the blood-brain barrier (BBB) has become a multidisciplinary task. In this two-volume handbook we try to cover all the different aspects that need to be considered to picture a modern image of this fascinating physiological phenomenon. During the past decade our knowledge on the BBB has largely improved, and as mentioned in our introduction, the focus has shifted from an endothelio-centric point of view to a more generalized perspective that includes additional cellular adjuncts like astrocytes, pericytes, microglial cells, and even stem cells. Insofar we prefer the term blood-brain interfaces when describing the various constituents that comprise this more holistic architecture of the BBB. It is useless to say that such an endeavor can never be complete, in particular when the different facets involve a host of structural, molecular, and cell biological aspects ranging from ontogenesis to the diseased BBB. The different chapters provide basic knowledge and state-of-the-art information that can be used for further perusal. Especially, the reference lists collected by the authors are treasures of their own, since they set pathways for additional in-depth studies. All contributors are experts in their field with a profound background in BBB research; and the editors are thankful for their enthusiasm which helped them to concentrate on their subjects outside the daily rush of paperwork. From this perspective, we hope this handbook will serve as a rich source of information for a wide audience, including graduate students, advanced undergraduates, and professionals. Our sincere thanks go to all contributors for contributing excellent and comprehensive chapters, to Mrs. Monika Birkelbach for secretarial assistance and to Dr. Andreas Sendtko and his colleagues at Wiley-VCH for continuous and encouraging support throughout the preparation of this handbook. Bochum, New York, Rochester January 2006


Rolf Dermietzel David C. Spray Maiken Nedergaard

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List of Contributors Joan Abbott Wolfson Centre for Age Related Diseases King’s College London Hodgkin Building Guy’s Campus London, SE1 1UL UK Barbara Aumayr Department of Neurosurgery Cleveland Clinic Foundation 9500 Euclid Avenue Cleveland, OH 44106 USA William A. Banks Veterans Affairs Medical Center 915 N. Grand Boulevard St. Louis, MO 63106 USA Ingo Bechmann Institute of Cell Biology and Neurobiology Charité University Hospital Berlin Schumannstrasse 20/21 10098 Berlin Germany

Richard Beliveau Département de Chimie-Biochimie Université du Québec à Montréal C.P. 8888, Succursale Centre-ville Montréal, H3C 3P8 Canada Nicholas Bodor Center for Drug Discovery University of Florida PO Box 100497 Gainesville, FL 32610-0497 USA Alejandra Bosco Department of Neuroscience Albert Einstein College of Medicine 1410 Pelham Parkway S Bronx, NY 10464 USA Peter Buchwald IVAX Research, Inc. 4400 Biscayne Boulevard Miami, FL 33137 USA Valérie Buée-Scherrer Faculté des Sciences Jean Perrin Université d’Artois SP 18, rue Jean Souvraz 62307 Lens France


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Laura M. A. Camassa Department of General and Environmental Physiology and Centre of Excellence in Comparative Genomics (CEGBA) University of Bari Via Amendola 165/A 70126 Bari Italy Roméo Cecchelli Faculté des Sciences Jean Perrin Université d’Artois SP 18, rue Jean Souvraz 62307 Lens France Tailoi Chan-Ling Department of Anatomy Institute for Biomedical Research University of Sydney Sydney, NSW 2006 Australia Yoon Kyung Choi Research Institute of Pharmaceutical Sciences and College of Pharmacy Seoul National University Seoul, 151-742 Korea Marilyn J. Cipolla Department of Neurology University of Vermont 89 Beaumont Avenue, Given C454 Burlington, VT 05405 USA Caroline Coisne Faculté des Sciences Jean Perrin Université d’Artois SP 18, rue Jean Souvraz 62307 Lens France

Luca Cucullo Department of Neurosurgery Cleveland Clinic Foundation 9500 Euclid Avenue Cleveland, OH 44106 USA Albertus (Bert) G. de Boer Blood-Brain Barrier Research Group Division of Pharmacology Leiden University Einsteinweg 55 2333 CC, Leiden The Netherlands Bénédicte Dehouck Faculté des Sciences Jean Perrin Université d’Artois SP 18, rue Jean Souvraz 62307 Lens France Lucie Dehouck Faculté des Sciences Jean Perrin Université d’Artois SP 18, rue Jean Souvraz 62307 Lens France Marie-Pierre Dehouck Faculté des Sciences Jean Perrin Université d’Artois SP 18, rue Jean Souvraz 62307 Lens France Michel Demeule Département de Chimie-Biochimie Université du Québec à Montréal C.P. 8888, Succursale Centre-ville Montréal, H3C 3P8 Canada


Rolf Dermietzel Department of Neuroanatomy and Molecular Brain Research Ruhr University Bochum Universitätsstrasse 150 44780 Bochum Germany Mahalia S. Desruisseaux Montefiore Medical Center Albert Einstein College of Medicine 1300 Morris Park Avenue Bronx, NY 10461 USA Gerald A. Dienel University of Arkansas for Medical Sciences Shorey Building 4301 W. Markham Street Little Rock, AR 72205 USA Jean-Bernard Dietrich Inserm U 575 Université Louis Pasteur 5, rue B. Pascal 67084 Strasbourg France Gerhard F. Ecker Department of Medicinal Chemistry University of Vienna Althanstrasse 14 1090 Vienna Austria Britta Engelhardt Theodor Kocher Institute University of Bern Freiestrasse 1 3012 Bern Switzerland

Pedro M. Faustmann Neuroanatomy/Mol. Brain Research Ruhr University Bochum Universitätsstrasse 150 44780 Bochum Germany Chantal Fournier Département de Chimie-Biochimie Université du Québec à Montréal C.P. 8888, Succursale Centre-ville Montréal, H3C 3P8 Canada Antonio Frigeri Department of General and Environmental Physiology University of Bari Via Amendola 165/A 70126 Bari Italy Pieter J. Gaillard to-BBB technologies BV Bio Science Park Leiden Gorlaeus Laboratories, LACDR Facilities-FCOL Einsteinweg 55 2333 CC, Leiden The Netherlands Hans-Joachim Galla Department of Biochemistry University of Münster Wilhelm-Klemm-Strasse 2 48149 Münster Germany

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Albert Gjedde Pathophysiology and Experimental Tomography Center and Center of Functionally Integrative Neuroscience Aarhus University Hospital 44 Norrebrogade 8000 Aarhus Denmark Steven Goldman Department of Neurology University of Rochester Medical Center 601 Elmwood Rd., Box 645 Rochester, NY 14642 USA Claus G. Haase Department of Neurology and Clinical Neurophysiology Knappschafts-Hospital Dorstener Strasse 151 45657 Recklinghausen Germany Kerri Hallene Department of Neurosurgery Cleveland Clinic Foundation 9500 Euclid Avenue Cleveland, OH 44106 USA Glenn I. Hatton Department of Cell Biology and Neuroscience University of California, Riverside 1208 Spieth Hall Riverside, CA 92521 USA

Damir Janigro Department of Neurosurgery Cleveland Clinic Foundation 9500 Euclid Avenue Cleveland, OH 44106 USA Julie Jodoin Département de Chimie-Biochimie Université du Québec à Montréal C.P. 8888, Succursale Centre-ville Montréal, H3C 3P8 Canada Abba J. Kastin Pennington Biomedical Research Center Blood-Brain Barrier Laboratory 6400 Perkins Road Baton Rouge, LA 70808 USA Kyu-Won Kim Research Institute of Pharmaceutical Sciences and College of Pharmacy Seoul National University Seoul, 151-742 Korea Sae-Won Kim Research Institute of Pharmaceutical Sciences and College of Pharmacy Seoul National University Seoul, 151-742 Korea Dorothee Krause Institute of Neuroanatomy and Molecular Brain Research Ruhr University Bochum Universitätsstrasse 150 44780 Bochum Germany


Christina Lilliehook Department of Neurology University of Rochester Medical Center 601 Elmwood Road, Box 645 Rochester, NY 14642 USA

Danny A. Milner Harvard Medical School Department of Pathology Brigham & Women’s Hospital 75 Francis Street, Amory 3 Boston, MA 02115 USA

Wee Shiong Lim Department of Geriatric Medicine Tan Tock Seng Hospital 11 Jalan Tan Tock Seng Singapore 308433 Republic of Singapore

Albert Moghrabi Centre de Cancérologie Charles Bruneau Université du Québec à Montréal 3175 Chemin Côte-Ste-Catherine Montréal, H3T 1C5 Canada

Christina Lohmann Discovery DMPK&BA AstraZeneca R&D Lund Scheelevägen 8 22187 Lund Sweden Sebastien Mambie Pacific Biomedical Research Center University of Hawaii 1993 East-West Road Honolulu, HI 96822 USA Frederic Mercier Pacific Biomedical Research Center University of Hawaii 1993 East-West Road Honolulu, HI 96822 USA Florence Miller Faculté des Sciences Jean Perrin Université d’Artois SP 18, rue Jean Souvraz 62307 Lens France

Maria G. Mola Department of General and Environmental Physiology and Centre of Excellence in Comparative Genomics (CEGBA) University of Bari Via Amendola 165/A 70126 Bari Italy Arshag D. Mooradian Division of Endocrinology Saint Louis University 1402 South Grand Blvd Saint Louis, MO 63104 USA Adam Mott Department of Immunology & Infectious Diseases Harvard School of Public Health 665 Huntington Avenue Boston, MA 02115 USA

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Maiken Nedergaard School of Medicine and Dentistry University of Rochester 601 Elmwood Avenue Rochester, NY 14642 USA

Emily Oby Department of Neurosurgery Cleveland Clinic Foundation 9500 Euclid Avenue Cleveland, OH 44106 USA

Grazia P. Nicchia Department of General and Environmental Physiology and Centre of Excellence in Comparative Genomics (CEGBA) University of Bari Via Amendola 165/A 70126 Bari Italy

Weihong Pan Pennington Biomedical Research Center Blood-Brain Barrier Laboratory 6400 Perkins Road Baton Rouge, LA 70808 USA

Beatrice Nico Department of Human Anatomy and Histology University of Bari Via Amendola 165/A 70126 Bari Italy Robert Nitsch Institute of Cell Biology and Neurobiology Charité University Hospital Berlin Schumannstrasse 20/21 10098 Berlin Germany Christian R. Noe Department of Medicinal Chemistry University of Vienna Althanstrasse 14 1090 Vienna Austria

Jeong Ae Park Research Institute of Pharmaceutical Sciences and College of Pharmacy Seoul National University Seoul, 151-742 Korea Josef Priller Department of Psychiatry Charité University Hospital Berlin Schumannstrasse 20/21 10098 Berlin Germany Markus Ramsauer Buchenrain 5 4106 Therwil Switzerland Ed Rapp Cleveland Medical Devices Inc. 4415 Euclid Avenue Cleveland, OH 44103 USA


Angelika Rappert Institute of Cell Biology and Neurobiology Charité University Hospital Berlin Schumannstrasse 20/21 10098 Berlin Germany Anthony Régina Département de Chimie-Biochimie Université du Québec à Montréal C.P. 8888, Succursale Centre-ville Montréal, H3C 3P8 Canada Bernhard Reuss Center for Anatomy University of Göttingen Kreuzbergring 36 37075 Göttingen Germany Domenico Ribatti Department of Human Anatomy and Histology University of Bari Via Amendola 165/A 70126 Bari Italy David C. Spray Department of Neuroscience Albert Einstein College of Medicine 1410 Pelham Parkway S Bronx, NY 10464 USA

Maria Svelto Department of General and Environmental Physiology and Centre of Excellence in Comparative Genomics (CEGBA) University of Bari Via Amendola 165/A 70126 Bari Italy Herbert B. Tanowitz Division of Infectious Diseases Albert Einstein College of Medicine 1300 Morris Park Avenue Bronx, NY 10461 USA Hong Tu Pennington Biomedical Research Center Blood-Brain Barrier Laboratory 6400 Perkins Road Baton Rouge, LA 70808 USA Sandra Turcotte Département de Chimie-Biochimie Université du Québec à Montréal C.P. 8888, Succursale Centre-ville Montréal, H3C 3P8 Canada Corine C. Visser Blood-Brain Barrier Research Group Division of Pharmacology Leiden University Einsteinweg 55 2333 CC, Leiden The Netherlands

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Christian Weidenfeller Department of Chemical and Biological Engineering University of Wisconsin-Madison 1415 Engineering Drive Madison, WI 53706 USA

Shulin Xiang Pennington Biomedical Research Center Blood-Brain Barrier Laboratory 6400 Perkins Road Baton Rouge, LA 70808 USA

Louis M. Weiss Division of Infectious Diseases Albert Einstein College of Medicine 1300 Morris Park Avenue Bronx, NY 10461 USA

Alla Zozulya Department of Pathology and Laboratory Medicine University of Wisconsin-Madison 1300 University Avenue Madison, WI 53706 USA

Hartwig Wolburg Institute of Pathology University of Tübingen Liebermeisterstrasse 8 72076 Tübingen Germany

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Introduction The Blood-Brain Barrier: An Integrated Concept Rolf Dermietzel, David C. Spray, and Maiken Nedergaard

Writing about the blood-brain barrier (BBB) these days has become a multidisciplinary enterprise. While the classic view formulated by the pioneering work of Ehrlich (1885) and Goldman (1913) was based on evidence that blood-borne substances were excluded from the brain, this concept was systematically reformulated over the past century, cumulating in the compartment concept put forward by Davson (1967) in which he redefined the BBB as the “sum of all bidirectional exchange processes which occur at the morphological blood-brain interfaces”. This definition has shifted the more or less endotheliocentric view to a more integrated concept that takes into account not only the bidirectionality of the exchange processes, but also the discovery that besides the endothelium additional components constitute integral parts of the barrier mechanisms. These additional components include the perivascular structural adjuncts such as pericytes, microglia and macrophages and the astroglial interface, interacting in an orchestrated fashion in order to achieve the “functional complex” that allows the transmission of metabolic and homeostatic information between the blood and the brain parenchyma and vice versa. In a former review on the BBB, one of the editors of this handbook described the function of this barrier as a dynamic homeostat (Dermietzel and Krause 1991) that regulates the interchange between the body and the brain border. By its nature, this homeostat is not simply constituted by the sum of its parts. The integration of all molecular and structural components and their concerted interplay at the critical locations gives rise to the “more” that we believe is the essence of synergism. Consequently, when conceptualizing this handbook we had to take into account the entire spectrum of disciplines that contribute to our modern understanding of the BBB. The picture we tried to paint cannot, of course, be complete and the reader may find missing a contribution that exactly covers his field of interest. In this case, we must direct the interested scholar to the list of references that are included in each of the articles and which may be well suited as a guideline for further deepening his knowledge. The collection of articles is thus structured in a way that reflects our understanding of the BBB as a dynamic homeostat. The first part deals with the ontoBlood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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Introduction – The Blood-Brain Barrier: An Integrated Concept

genesis of brain vasculature and begins with a basic article written by Britta Engelhardt that not only describes the maturation of the BBB and its structural substrate, but also goes into details regarding molecular aspects of the developmental processes. What we have learned during recent years, especially through the work by the late Werner Risau and his coworkers, is that when the BBB is first established it is by no means a static structure, but rather a dynamic construct that requires permanent feeding by growth and differentiation factors in order to maintain and elaborate its complex machinery as an exquisite barrier. As we now know, the microvasculature is not uniformly tight throughout the brain, but is a composite building that contains segments of leakiness integrated into the mass of tight vessels. These leaky segments prevail at sites where a requirement for differential exchange between the blood site and the brain site (or vice versa) exists, i.e. neurohaemal regions which entail neurosecretory and neurosensory areas for controlling specific parameters of the body fluid. An understanding of how the brain manages to keep leaky segments maintained in an embedment of tight vessels will provide a key to the understanding of BBB development. The article by Jeong Ae Park, Yoon Kyung Choi, Sae-Won Kim and Kyu-Won Kim focuses on some of these factors with emphasis on hypoxia inducible factor (HIF), VEGF and other neuroglia-derived factors. It describes the main sources of these potent humoral factors and their effects during angiogenesis on the developing brain including aspects for barriergenesis. An extension of the discussion on humoral regulation of brain angiogenesis is provided by Christina Lilliehook and Steven Goldman. Their focus is on a rather new and exciting field of BBB research which considers the brain microvasculature as a niche that conditions brain stem cells for further lineaging. The observation that hippocampal stem cells before they integrate into the granular cells of the dentate gyrus associate with cerebral capillaries has made this niche a favorable subject for unravelling the conditioning mechanisms taking place at the sites of contact. With the concept of a vascular niche a further aspect of the BBB emerges, namely that brain vessels not only provide targets for autocrine and paracrine regulation to maintain their own degree of differentiation, but constitute a putative source of fostering brain stem cells. Part II is devoted to the different structural components that define the BBB. Without surprise, this part begins with an account on our recent understanding of the structural and molecular complement of the cerebral endothelium written by Hartwig Wolburg. The endothelium plays a crucial role in separating the blood from the two major fluid compartments of the brain: the interstitial cerebral compartment and the cerebrospinal compartment. As mentioned above, this separation entails a constitutive dynamic component which is responsive to the actual metabolic situation on both sites of the barrier. The chapter on the endothelium is not intended to give a complete survey covering all the various carrier and transport mechanisms taking place at the endothelial frontier. Such an encyclopedic detailed overview is outside the scope of this handbook; and we refer here to recent reviews and monographs that deal with these particular


aspects of the BBB (Pardridge 1993; Paulson et al. 1999; Nag 2003). Rather, this chapter presents the major facts and our recent knowledge regarding the two major components of the barrier: its structural and metabolic substrates. The subsequent articles in this Section concentrate on the cellular adjuncts that surround the vessel wall and should be highlighted together in our conceptual context. Pericytes, presented by Markus Ramsauer, brain macrophages by Frederic Mercier, Sebastien Mambie and Glenn Hatton and microglial cells by Ingo Bechmann, Angelika Rappert, Joseph Priller and Robert Nitsch are all cellular components which permanently or transiently settle in the perivascular space. Cerebral pericytes have long been neglected by the BBB community although already described in the last third of the 19th century by Rouget. In spite of the fact that brain and retinal capillaries possess the highest density of pericytes per endothelial cell (about 1 : 3), their functional properties and morphological phenotype are still difficult to describe. They seem to be involved in microvascular perfusion by reciprocal contraction as well as in immune responses, a property that has god-fathered them as a “second line of defense” by their ability to enfold macrophagic activity under challenging conditions. Recent discoveries feature pericytes as key players of BBB differentiation, together with astrocytes and regulators of endothelial growth as well as stabilizers of the vascular wall. Their potential to differentiate into other mesenchymal cell types makes them a versatile cellular pool with progenitor features for macrophages and even endothelial cells. Macrophages, which are of mononuclear origin, reside in the whole CNS vasculature. Together with microglial cells they are responsible for the immune response of the brain and their mostly perivascular association makes them part of the BBB complex. Macrophages are potent producers of extracellular matrix molecules, growth factors and cytokines and are thought to play key roles in regulating glial and neuronal cell function. In contrast to microglial cells which have been detected by recent efforts (Nimmerjahn et al. 2005) to be primarily resident even when BBB injury commences, macrophages seem to be more migratory, patrolling the brain parenchyma and cleansing it from debris after injury or inflammatory attacks. A recent concept pointed out by the paper of Frederic Mercier, Sebastien Mambie and Glenn I. Hatton calls attention to the presumed function of macrophages as partners in the neurogenic niche, which is supposed to play a crucial role in neural precusor or stem cell priming. Microglial cells are by far the most abundant cellular entity (about 15% of brain cells) involved in the immune response of the CNS. They serve as sensors for pathologic events in the brain tissue and reside in the brain parenchyma and in perivascular and juxtavascular positions. Here, besides their paramount function as executors of the innate and adaptive immune responses of the brain tissue, they seem to function in concert with perivascular macrophages in regulating the tightness of the barrier through chemokine secretion. Both cellular entities, perivascular macrophages and microglial cells, perform fundamental functions in the regulation of the leakiness of the BBB under inflammatory conditions, an issue that is addressed in more detail in part VII of the handbook.

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The following two articles by Joan Abbott and the group of Antonio Frigeri (Grazia P. Nicchia, Batrice Nico, Laura M.A. Camassa, Maria G. Mola, Domenica Ribatti, David C. Spray, Alejandra Bosco, Maria Svelto and Antonio Frigeri) concentrate on the astroglial surrounding of the blood-brain interface. Since the work by Goldstein and Betz (1986) and later by Janzer and Raff (1987), the astroglia and in particular the perivascular endfeet have been considered to be crucial elements for maintaining the BBB complex. Astrocytes not only participate in regulating transport through the endothelium and provide trophic support for the tightness of the endothelium, but they also provide the essential link for vascular-neuronal signaling. Because of their ideal strategical position and their molecular and structural polarization, the astroglia are destined to link the neuronal site with the vascular bed of the brain parenchyma and serve as a pathway for metabolic and ionic transfer. Most importantly, astrocytes seem to represent the most dynamic part of the BBB complex, since they are able to respond to neuronal activity and transmit signals to the blood front to regulate local perfusion. Thus, the astroglia are an essential part of an integrated concept of the BBB, not only in terms of their morphogenetic capabilities, but also as transformers of neuronal activation into BBB receptive signals. Part III is devoted to hormonal and enzymatic control of the brain vessels. The initial article by Bernhard Reuss centers on the role of fibroblasts growth factors (FGFs) in regard to their influence on growth and differentiation of brain microvascular endothelium, and thus their involvement in establishing, maintaining and restoring the BBB. In particular, FGF-2 has come into focus as an important cofactor together with secretory products of astrocytes (TGF-b1, GDNF) involved in the induction of certain specific barrier properties of brain microvessels. Also, the effect of FGF-2 to influence the synthesis and phosphorylation of the intermediate filament protein GFAP of astrocytes, which has been proven to be important for inducing the BBB phenotype, is covered here, to provide insight of the importance of the FGF family for the stabilization and preservation of BBB features. This issue of factors that influence the functional properties of the BBB by humoral and enzymatic inputs is further considered by four articles. First, the contribution of Weihing Pan, Shulin Xiang, Hong Tu and Abba Kastin presents a general overview on the interaction of cytokines with the BBB. In particular, the mechanism of cytokine transport across the endothelial cells is described, as is their action on these cells, which results in altered endothelial function, cytotoxicity or cell proliferation. This article is followed by a description of insulin transport through the barrier and the effect of insulin on the BBB in concert with proinflammatory cytokines, authored by William Banks and Wee Shion Lim. It also addresses the important clinical issue of insulin resistance and its impact on the inflammatory susceptibility of the brain vasculature, an issue that is of profound socio-economic importance in view of the increasing number of obese people. The reviews on humoral effects on the BBB is completed by Jean Bernard Dietrich, who gives a detailed account of the interaction of glucocorticoid hormones and estrogens on the endothelium of the BBB. The focus of this


contribution lies on the influence of glucocorticoids and estrogens with regard to their expression of endothelial adhesion molecules that are pivotal for the transendothelial migration of inflammatory cells through the BBB. In this context, their use as therapeutic tools for the treatment of autoimmune diseases is discussed. Part III on hormone and enzyme interaction ends with the chapter of Dorothee Krause and Christina Lohmann on metalloproteinases and the brain microvasculature. Besides the structural sealing of the BBB endothelium through tight junctions and its complement of specific transporters and carriers, the vascular wall of brain vessels is endowed with a battery of enzymes constituting a kind of “enzymatic barrier” to the passage of peptides across the BBB. These enzymes are proteinases and have been detected in variable amounts at the BBB. Among them are the metalloproteinases, which are involved in the cleavage of peripheral peptides as well as centrally released peptides, in remodelling the extracellular matrix during angiogenesis and in facilitating perivascular penetration of emigrating blood-borne cells, including tumor cells. The complexity of the metalloproteinases (MPs), and in particular the matrix bound subgroup (MMPs), for the function of the BBB are thoroughly reviewed in this paper. Part IV centers on different approaches that have been taken to establish in vitro systems for culturing the BBB complex. It further includes a strategy that has recently been developed aimed to predict BBB properties by in silico approaches. In vitro models are of paramount importance to test the transport of drugs through the BBB. The use of alternate tissue culture models are helpful in some ways, but can only be regarded as approximative approaches. Thus, considerable effort has been channeled to develop reliable culture systems to mimic the BBB in vitro. The chapter by Romeo Cecchelli, Caroline Coisne, Lucie Dehouck, Florence Miller, Marie-Pierre Dehouck, Valérie Buée-Scherrer and Bénédicte Dehouck gives an overview on our present knowledge of in vitro BBB models that at least fulfil some of the essential criteria that are required for a well differentiated BBB endothelium. In extension of this article, Alla Zozulya, Christian Weidenfeller and Hans-Joachim Galla describe their specific approach to utilize a coculture system treated with hydrocortisone to achieve monolayers with high endothelial resistance, the key feature of a tight barrier. Both articles provide a comprehensive source of information for those who are interested in model systems of the BBB. The chapter by Luca Cucullo, Emily Oby, Kerri Hallene, Barbara Aumayr, Ed Rapp and Damir Janigro introduces advanced models that take into account the three-dimensional structure of the vascular tube including shear-factors applied on the endothelium by fluid flow. This dynamic in vitro model of BBB (DIV-BBB) and its newly designed model (NDIV-BBB) display the most advanced ex vivo approach to the in vivo BBB situation with the advantage of large upscaling, an obligatory requirement for industrial application. Gerhard Eckert and Christian Noe introduce the readers to the new world of in silico screening of drugs (pharmacoinformatics) with respect to their ability to penetrate the BBB. These in silico methods gain increasing interest in order to economize the process of standard high-throughput screening. The applica-

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bility of computational methods on the BBB is a novel promising strategy that may allow discovery of target families and presumably novel bioactive molecules. The fact that the permeation of the BBB is a multifactorial process necessitates advanced computational methods for modelling approaches and opens new perspectives in BBB drug research. Part V completes consideration of pharmacological aspects of the BBB, and gives access to some fundamental issues of BBB pharmacology. Sandra Turcotte, Michel Demeule, Anthony Régina, Chantal Fournier, Julie Jodoin, Albert Moghrabi and Richard Béliveau introduce one of the most relevant systems that prevents significant accumulation of many hydrophobic molecules and drugs in the brain: the multidrug resistance transporter P-glycoprotein (P-gp). This efflux transporter is a member of the ATP-binding cassette group of transporters (ABC), which represent the largest family of transmembrane proteins. The existence of the P-gp at the BBB is one of the main causes of failure in chemotherapy, because of its ability to translocate xenobiotics against a concentration gradient across the plasma membrane. Thus, the P-gp plays an important role in brain protection at the BBB site, but by its nature provides a considerable hindrance for successful treatment of a variety of brain diseases. Nicolas Bodor and Peter Buchwald provide a survey on general aspects of targeting neuropharmacologicals by chemical delivery systems (CDSs). CDSs, as the authors state, represent a rational drug design approach that exploits sequential metabolism not only to deliver, but also to specifically target drugs to their site of action. The authors present a spectrum of approaches intended to deliver drugs, particularly bioactive peptides, through CDSs to the brain tissue. The chapter by Pieter J. Gaillard, Corine C. Visser and Albertus G. de Boer is centered on certain delivery systems, which include the transferrin system, the insulin receptor, the low-density lipoproteins I and II (LRP1, LRP2) and the diphtheria toxin receptor. The authors present the enormous opportunity that these systems offer for the successful delivery of drugs to the brain, but also enumerate the pitfalls that these systems face and their still limited applicability in clinical therapeutics. Two major chapters herald part VI, both of which deal with vascular perfusion. Albert Gjedde introduces the field of blood-brain transfer and metabolism of oxygen, a field that sets the basics for functional imaging covered by the article of Gerald A. Dienel. The rationale to include these chapters within this handbook on the BBB is multi-faceted. As quoted by A. Gjedde, delivery of oxygen to brain tissue differs in major respects from the delivery of oxygen to other tissues. This is not a direct consequence of the properties of the BBB in its narrow sense, but reflects the specific demands that the vascular system of the brain must fulfil in order to satisfy the energetic requirements of the working brain. “The absent recruitment of capillaries in states of activation of neurons as well as the general principle of topographic arrangement of the vessels” account for the differences of brain with regard to other tissues. This statement by A. Gjedde pin-points the morphological and physiological specificity of the brain microvasculature, a key feature in the context of an integrated concept of the BBB. This is followed directly by the chapter of Gerald Dienel, which covers the


entire spectrum of modern brain imaging and provides a thorough overview of the basic physiological features on which modern brain imaging is founded. The “take-home message” of this chapter is that blood flow, metabolism and cellular function are inseparable aspects of brain activities and the responsiveness of the BBB to neuronal activity represents a hallmark in coupling both sites of the active brain: blood flow and brain work. The last and most extensive part VII includes a collection of articles on clinical afflictions of the BBB. It starts with a chapter on the impact of inflammation on the BBB by Pedro Faustmann’s and Claus Haase’s contribution on inflammatory responses of the blood-brain interface. The brain has long been considered to be an immunoprivileged part of the body. Under pathological conditions such as inflammation, trauma and neurodegeneration blood-borne cells immigrate into the CNS and changes of the permeability of the BBB occur. The mechanisms how these cells enter the brain and which proteins in the cerebrospinal fluid are disease-related are key issues, which represent central questions currently being addressed in neurology, neuropathology and neuroimmunology. The sections on inflammatory attacks on the BBB is followed by Marilyn J. Cipolla’s chapter on stroke that summarizes the pathophysiological sequelae to the event of a stroke, i.e. development of brain edema, cellular regulation of cerebrovascular permeability, effects of stroke on the cytoskeleton of brain endothelium and reperfusion injury, just to mention a few highlights of this article. A further important clinical issue is covered by the article of Arshag D. Mooradian, which is centered on diabetes and its consequences for the BBB. “The CNS complications of diabetes have not widely appreciated, because of the most overt complication, namely stroke”, states A. Mooradian, pointing to the more subtle effects on the CNS that extends beyond clinically appreciated cerebrovascular accidents. These include: alterations in cerebral microvessels with poor autoregulation and blood distribution, altered BBB function, neurochemical changes, alterations in neurotransmitter receptor activity and contributing factors such as hypoglycemic reactions. All these changes in the CNS are discussed from the perspective of BBB function. The chapter by a group of parasitologists contributing to the chapter by Danny A. Milner (Mahalia S. Desruisseaux, Louis M. Weiss, Herbert B. Tanowitz and Adam Moss) focuses on a group of infections with global impact: human parasitic diseases. The involvement of the BBB during parasitic infections has been elaborated in this conceptual context for the first time in this handbook. The chapter by Milner et al. offers a taxonomic guideline for those readers who are interested in parasitic infections which affect, disrupt and/or destroy the BBB. From a clinical point of view, these infections are of great relevance since they are more commonly fatal than parasitic diseases which do not destroy the BBB. The final article by Tailoi Chang-Ling introduces the current understanding of the blood-retina interfaces. It describes both the features the retinal interface shares in common with the CNS blood vessels as well as its specific structural features. The unique morphology of the retina as an exposed part of the brain

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and the transparency of the ocular media has made it an exceptionally accessible target not only for in vivo detection of the vascular physiology but also for monitoring cardiovascular pathology, including changes to arterioles and venular walls due to arteriosclerosis and diabetic retinopathy. We feel that this is an appropriate last chapter, insofar as the article recapitulates many of the concepts regarding both healthy and diseased BBB that are encountered during the excursion through this handbook.

References

Davson, H. 1967, Physiology of the Cerebrospinal Fluid, Churchill, London. Dermietzel, R., Krause, D. 1991, Molecular anatomy of the blood-brain barrier as defined by immunocytochemistry, Int. Rev. Cytol. 127, 57–103. Ehrlich, P. 1885, Das Sauerstoffbedürfnis des Organismus. Eine farbanalytische Studie Hirschwald, Berlin. Goldmann, E. 1913, Vitalfärbung am Zentralnervensystem. Abh. K. Preuss. Akad. Wiss. Phys. Med. 1, 1–60. Goldstein, G. W., Betz, A. L. 1986, The blood-brain barrier. Sci. Am. 255, 74–83. Janzer, R. C., Raff, M. C. 1987, Astrocytes induce blood-brain barrier properties in endothelial cells. Nature 325, 253–257. Nag, S. 2003, The Blood-Brain Barrier, Humana Press, Totowa. Nimmerjahn, A., Kirchhoff, F., Helmchen, F. 2005, Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308, 1314–1318. Pardrige, W. M. 1993, The Blood-Brain Barrier: Cellular and Molecular Biology, Raven Press, New York. Paulson, O. B., Moos Knudsen, G., Moos, T. 1999, Blood-Brain Barrier Systems Munksgaard, Copenhagen.

Part I Ontogeny of the Blood-Brain Barrier


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1 Development of the Blood-Brain Interface Britta Engelhardt

1.1 Introduction

The blood-brain barrier (BBB) is composed of a continuous layer of highly specialized vascular endothelial cells. The BBB maintains central nervous system (CNS) homeostasis by preventing the entry of substances that might disturb proper function of the neurons. Uncontrolled paracellular diffusion of hydrophilic molecules into the CNS is inhibited by a complex network of tight junctions between the endothelial cells. Transcellular passage of molecules into the CNS is prohibited by the lack of endothelial fenestrae and an extremely low pinocytotic activity of these endothelial cells. In order to meet the high metabolic needs of the neurons “behind” the barrier in spite of its restrictive capacity, specific transport systems are selectively expressed in the CNS microvascular endothelial cells, which mediate the directed transport of nutrients from the blood into the CNS or the removal of toxic metabolites out of the CNS. Recent years have dramatically advanced our knowledge about the growth factors and their receptors specifically acting on the developing vascular endothelium including the CNS vasculature. Despite this increased knowledge, all we know about the molecules involved in inducing BBB characteristics in endothelial cells during embryogenesis and maintaining them in the adult is that they are provided by the CNS microenvironment. In this chapter, the current knowledge of the cellular and molecular mechanisms involved in the induction, development and maintenance of the BBB is summarized.

1.2 Pioneering Research on the Blood-Brain Barrier

Discovery of the BBB is usually ascribed to the work of the German immunologist Paul Ehrlich. However, although he discovered in the 1880s that certain dyes, when injected into the vascular system of experimental animals, were rapidly taken up by all tissues with the exception of the brain and spinal cord [1], Blood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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1 Development of the Blood-Brain Interface

he did not asign this phenomenon to the existence of a barrier between the blood and the brain but rather interpreted his findings as a lack of affinity of the nervous system for these dyes. Only afterwards, when Edwin E. Goldman, an associate of Ehrlich, showed that the very same dyes, when injected into the cerebrospinal fluid, stained brain and spinal cord tissue but not any tissue outside of the CNS [2], was it concluded that these dyes were prevented from either getting access from the blood to the CNS or vice versa from the CNS to the blood circulation, suggesting the existence of a vascular barrier between the blood and the CNS [3]. The term “blood-brain barrier” was coined by Lewandowsky [4]. Based on the observation made by him and others that neurotoxic agents affected brain function solely when directly injected into the brain, but not when injected into the vascular system, he concluded the existence of a BBB [4, 5]. Until 1967, it was not clear whether the structural basis for the BBB was located at the level of endothelial or rather glial cells. Thomas Reese and Morris Karnowsky [6] were able to show by means of high resolution electron microscopy and the development of electron-dense tracers that intravenously injected horseradish peroxidase (HRP) was able to penetrate between endothelial cells in heart and muscle but not in the brain, where its diffusion was blocked at the level of tight junctions between the brain endothelial cells [6, 7]. Interestingly, exogenous peroxidase was also localized in some micropinocytotic vesicles within endothelial cells but none was found beyond the vascular endothelium, suggesting that vesicular HRP also did not reach the brain. It should be noted that, with the exception of the elasmobranch fishes (which like many invertebrates [8] have a BBB at the level of glial cells [9]), in all vertebrates the BBB is localized at the level of the endothelial tight junctions. Within the CNS there are some areas with neurohemal or neurosecretory functions (i.e. their neurons monitor hormonal stimuli and other substances within the blood or secrete neuroendocrines into the blood) which lack a vascular barrier [10]. As they are strategically localized in the midline of the ventricular system, they are collectively referred to as circumventricular organs (CVOs). The capillaries within the CVOs are fenestrated, allowing free diffusion of proteins and solutes between the blood and the CVOs. A barrier is, however, established by a complex network of tight junctions connecting specialized ependymal cells (tanycytes) at the border of the CVOs [10, 11]. Yet another structure, where endothelial cells do not form a barrier within the CNS, is the choroid plexus [12]. The choroid plexus is a villous structure consisting of an extensive capillary network enclosed by a single layer of cuboidal epithelium. It extends from the ventricular surface into the lumen of the ventricles. Its major known function is the secretion of cerebrospinal fluid. The choroid plexus capillaries are fenestrated like those in the CVOs, while tight junctions surround the apical regions of the choroid plexus epithelium, forming the blood-cerebrospinal fluid barrier. Resembling the situation within the choroid plexus, barriers in the vertebrate retina, an external part of the brain, show a similar organization. Whereas the intraretinal blood vessels form a blood-retina barrier (BRB), the vessels of the choroidea are fenestrated and the barrier is formed by the retinal pigment epithelium.

1.3 The Mature Blood-Brain Interface


The fully differentiated BBB is composed of at least four different cell types, which all contribute to the regulation of the BBB. Embedded within the basal membrane of the highly specialized endothelial cells, a high number of pericytes can be found (about one pericyte per 2–3 endothelial cells). More than 99% of the abluminal surface of the CNS capillaries is invested by astrocytic endfeet [13] leaving only a small distance of 20 nm between the astrocytic foot process and the endothelial cell surfaces. It should be noted that nerve terminals derived from intracerebral and extracerebral neurons have also been reported in close proximity to brain endothelial cells [14, 15]. The space between the endothelial cells/pericytes and the astrocytes therefore forms the interface between the blood and the brain. Interestingly, in this strategic location, cells of the immune system can be found, namely perivascular macrophages or dendritic cells. Whether they have a direct influence in the proper function of the BBB remains to be investigated. Although the endothelial cells form the barrier proper, the interaction with the abovementioned cells adjacent to the endothelium is required for a proper barrier function. The most prominent feature of the BBB is the presence of complex tight junctions between CNS endothelial cells, which establish a high electrical resistance across the endothelial barrier (about 2000 X cm–2) (see also Chapter 4) [16]. This seems to be due to the fact that, unlike cell junctions found between endothelial cells in peripheral blood vessels, the cell junctions in the brain are more extensive and seem to form an unbroken belt between the endothelial cells. Therefore, measurement of the transelectrical resistance (TER) across brain endothelium has become a well accepted criterion for BBB maturity or of barrier characteristics established by brain endothelial cells in vitro. BBB tight junctions can morphologically be distinguished from tight junctions between endothelial cells in the periphery, especially by freeze fracture electron microscopy. Whereas in BBB endothelial cells tight junction particles are preferentially associated with the protoplasmic face (the P-face), in nonbarrier endothelial cells tight junctional particles clearly predominate at the exocytoplasmic face (the E-face) [17, 18]. Interestingly during BBB development in the rat, it was observed that, besides the complexity of tight junctions, their P-face association increases [19]. As cultured BBB endothelial cells demonstrate a correlation of the P-face association of tight junction particles with the barrier function of the BBB endothelium [20], P-face association of tight junctions might be a measure for BBB maturation, at least in mammals. It should be noted that, in submammalian species, brain endothelial cells generally show tight junctions with high P-face association [21] and during BBB development in the chicken only tight junction/strand complexity is observed to be upregulated [19, 22, 23]. As elegant freeze-fracture immunogold staining studies have demonstrated, the localization of transmembrane tight junction proteins within the particle strands on P- and E-face freeze-fracture preparations [24–26], particle distribution in freeze-fracture

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analysis is most likely a function of the cytoplasmic anchoring of transmembrane tight junction proteins, which might be different in BBB and nonBBB endothelia [20]. P-face association of tight junction particles has become an accepted morphological criterion of endothelial barrier properties in mammals. It remains to be shown whether the above mentioned species differences in tight junction/morphology correlate with different barrier properties. Occludin was the first integral membrane protein found to be exclusively localized within tight junctions, including the BBB (Fig. 1.1) [27]. However, mice carrying a null mutation in the occludin gene are viable and develop morphologically normal tight junctions in most tissues, including the brain [28]. Thus, although occludin localizes to tight junction particles in freeze-fracture preparations, it is not essential for proper tight junction formation. In contrast to occludin, the claudins, which exhibit no sequence homology to occludin, comprise an entire gene family of integral membrane tight junction proteins, with more than 20 members to date; and they have been shown to be sufficient for the formation of tight junction strands [25]. Claudins are not randomly distributed throughout the tissues. Besides the endothelial specific claudin-5, claudin-3 was shown to be localized in endothelial tight junction in the CNS of mice and man [29]. Additionally, claudin-12 has been described in CNS endothelium [30]. The presence of claudin-1 in brain endothelial cells is still controversial (see [29, 31, 32]). Part of the confusion has been caused by the use of an anti-claudin-1 antibody that was subsequently shown to cross-react with claudin-3. The present

Fig. 1.1 BBB tight junctions. Schematic overview of the molecular composition of the cell-to-cell junctions between CNS microvascular endothelial cells and their putative linkage to the endothelial cytoskeleton.


availability of specific antibodies and molecular biology techniques should help to clarify the exact claudin makeup of BBB tight junctions in the near future. With regard to BBB biology it is exciting that, in the absence of occludin, transfection of claudin-3 into fibroblasts (which lack tight junctions) induces P-face-associated (i.e. BBB-like) tight junctions [33]. In contrast, transfection of fibroblasts with the endothelial cell-specific claudin-5 induces E-face-associated tight junctions in the absence of occludin [24]. These observations suggest that different claudins induce structurally different tight junctions and that at the BBB claudin-3 and claudin-5 might be responsible for the presence of P-face- and E-face-associated tight junctions particles, respectively. That individual claudins within the BBB tight junctions fulfill specific functions regarding the regulation of the paracellular permeability has been shown by the establishment of mice deficient for claudin-5, which die as neonates about 10 h after birth due to a size-selective loosening of the BBB for molecules smaller than 800 Da [30]. Thus, each claudin seems to regulate the diffusion of a group of molecules of a certain size. Additionally, members of the immunoglobulin supergene family have been described to be localized within tight junctions, including those of the BBB. Specifically, junctional adhesion molecule A (JAM-A [34]) and the endothelial cell-selective adhesion molecule (ESAM [35]) have been investigated in this context. JAM-A deficient mice have been established and described to be viable [36]. JAM-A deficient mice did not demonstrate significant alterations in organ development or morphology nor in vascular patterning or permeability, suggesting that JAM-A is not essential for proper BBB development. However, JAM-A has been demonstrated to regulate endothelial cell motility probably by providing an anchor to the cytoskeleton and might therefore fulfill a regulatory function at the BBB [37]. The functional involvement of ESAM in BBB tight junctions remains to be established. The integral membrane proteins of the tight junctions, including those of the BBB [38], are linked to the cytoskeleton via cytoplasmic multidomain scaffolding proteins of the peripheral membrane associated guanylate kinase (MAGUK) family, such as ZO-1, ZO-2 and ZO-3 (Fig. 1.1; for reviews, see [39, 40]). Besides providing the cytoskeletal anchorage of transmembrane tight junction proteins, the MAGUKs seem to be important for the correct spacial distribution of the individual transmembrane tight junction proteins within the tight junction by binding their cytoplasmic domains via different domains [41, 42]. As ZOs have been reported to shuttle between tight junctions and the nucleus, they may also regulate gene expression at the BBB. In cerebral endothelial cells, nonoccluding adherens junctions are found intermingled with tight junctions [43]. In adherens junctions, the endothelial specific integral membrane protein VE-cadherin [44] is linked to the cytoskeleton via catenins, which belong to the family of armadillo proteins (Fig. 1.1) [45]. In endothelial cells, expression and localization of b-catenin, c-catenin and p120cas have been described to be crucial for the functional state of adherens junctions, including those in the brain [46, 47]. VE-cadherin has been shown to mediate contact inhibition of endothelial cell growth. The contribution of VE-cadherin

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and the catenins in maintaining the integrity of the BBB remains to be investigated, as expression of VE-cadherin and the catenins in the mature BBB was reported to be low [48, 49]. Finally, PECAM-1 has been demonstrated to localize to endothelial cell contacts outside of either tight junctions or adherens junctions, including the brain. In mice deficient for PECAM-1, however, no primary defect in BBB integrity has been reported [50]. However, in the case of chronic inflammation (such as experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis), mice deficient for PECAM-1 demonstrated earlier extravasation of mononuclear cells as compared to wild-type control animals [51]. Furthermore, PECAM-1 deficient mice showed a prolonged and exaggerated vascular permeability of CNS vessels during EAE, suggesting that PECAM-1 is a negative regulator of leukocyte migration across CNS microvessels and a positive regulator of BBB maintenance. As indicated before, the junctional systems are connected to the endothelial cytoskeleton. The importance of the cytoskeleton in the establishment and maintenance of the BBB has become evident by mice lacking the actin-binding protein dystrophin. These so called mdx mice exhibit an increase in brain vascular permeability due to the disorganization of the a-actin cytoskeleton in endothelial cells and astrocytes, which leads to the altered subcellular localization of junctional proteins in the endothelium as well as the water channel aquaporin4 (AQP4) in astrocytic endfeet [52]. The physical BBB established by specialized tight junctions and a low number of pinocytotic vesicles implies the necessity for specific transport systems which can ensure the transport of “nutrition” to the brain parenchyma. This biochemical BBB is established by transport systems of the BBB which can be grouped into three types: carrier-mediated transport, active efflux transport and receptormediated transport. Members of each family have been cloned at the BBB [53]. The glucose transporter Glut-1, belonging to the carrier-mediated transporters, is specifically expressed on brain endothelial cells with higher localization at the abluminal versus the luminal membrane [54]. The receptor-mediated transporters of the BBB that have been cloned to date include the leptin receptor (OBR) and the transferrin receptor. The transferrin receptor can be observed on endothelial cells forming a BBB but not on those within the choroid plexus or the CVOs [55]. From the group of active efflux transporters, expression of P-glycoprotein was shown to be required for the BBB and seems to ensure the rapid removal of ingested toxic, lipophilic metabolites from the neuroectoderm [56]. In addition to the physical and biochemical barrier, the specialized endothelial cells of the BBB provide a metabolic barrier by the expression of a number of enzymes which modify lipophilic endogenous and exogenous molecules, which otherwise could bypass the physical or biochemical barrier and negatively affect neuronal function (for detailed reviews, see [53, 57, 58]). Although the above-described characteristics have originally been described solely for one particular segment of the vascular tree (namely the capillaries), more recently the term BBB has been used more widely, especially by immunologists to describe the unique characteristics of the CNS microvessels in the context

1.4 Development of the CNS Vasculature

of CNS inflammation and leukocyte extravasation across the BBB. The assumption that the unique characteristics of CNS capillary endothelial cells extend to the level of the endothelial cells lining the postcapillary venules, the vascular segment where leukocyte extravasation takes place, bears some danger as it is well known that there are functional and molecular differences between endothelial cells in different segments of the vascular tree. However, as both the unique molecular composition of tight junctions and the expression of several of the BBB specific transport systems have been described in microvascular endothelial cells of the CNS, the precise vascular localization of the functional term BBB might be extended beyond the capillary segments to the CNS microvessels [29, 59]. In any case, endothelial cells comprising the barrier characteristics in the CNS are unique and clearly distinguishable from any other endothelial cell in the body. The determination of how much variability there is in BBB characteristics along the vascular tree within the CNS awaits analysis. To understand how brain endothelial cells acquire the unique features of the BBB, it is therefore crucial to understand the development of the brain vasculature.


During vertebrate embryogenesis, the development of the CNS vasculature begins when angioblasts, which originate from the lateral splanchnic mesoderm, enter the head region and form the perineural vascular plexus by de novo formation of blood vessels, a process called vasculogenesis. In a 2-day-old chick embryo and a 9-day-old rodent embryo, the perineural vascular plexus covers the entire surface of the neural tube [60–62]. None of the precursors that form the perineural vascular plexus nor the intraneural vessels are derived from the neuroectoderm [63, 64]. Thus, the vascular system within the CNS does not develop by vasculogenesis. Rather, vascular sprouts from the perineural plexus invade the proliferating neuroectoderm at day 4 of embryonic development in the chicken and at day 10 in rodents [60]. This mechanism whereby new vessels are formed from pre-existing vessels is called angiogenesis [61, 64, 65]. During brain angiogenesis, sprouting vessels grow radially into the neuroectodermal tissue, elongate, give rise to manifold branches and finally anastomose with adjacent sprouts, forming an undifferentiated network of capillaries near the ventricular zone of the developing brain [60, 66]. The onset of angiogenesis in the brain occurs at precisely reproducible stages of embryonic development (i.e. day 4 in chicken, day 10 in rodents) and follows a stereotypical temporal and spatial pattern, with a peak of angiogenic activity in early postnatal stages, establishing a reproducible pattern of cortical vascularization in mammals [60, 67]. The precise spatio-temporal orchestration of vascular development in the brain prompted Werner Risau in the 1980s to hypothesize that a paracrine mechanism operates by which angiogenic factors produced by the embryonic brain could activate specific receptors expressed on endothelial cells, thus lead-

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ing to migration and proliferation of these cells. The first growth factors with angiogenic activities isolated from embryonic chicken brain were identified as acidic (aFGF) and basic fibroblast growth factors (bFGF) [68]. Although aFGF and bFGF (FGF-1, FGF-2) meet the requirements to be potent inducers of endothelial cell proliferation in vitro and of angiogenesis in vivo, developmental studies on their expression pattern in the brain and on their corresponding receptor on endothelial cells did not correlate well with the spatio-temporal pattern of brain angiogenesis [69–71]. In fact, expression of FGFs remains high, even in the adult brain when angiogenesis has ceased [72–74]. After vascular endothelial growth factor (VEGF) was identified, it became clear that this was the candidate paracrine factor stimulating specifically endothelial cell proliferation and sprouting via its high affinity receptors VEGF receptor 1 (VEGFR1, flt-1) and VEGF receptor 2 (VEGFR2, flk-1/KDR) [75]. The analysis of null or conditional alleles for VEGF and its endothelial tyrosine kinase receptors has since demonstrated that VEGF is required for the formation, remodeling and survival of embryonic and early postnatal blood vessels, including the brain, in a dose-dependent manner (for a summary, see [76]). In the brain, VEGF is produced by cells in the ventricular layer forming a concentration gradient decreasing towards the neuroectodermal surface. The vessels originating from the perineural plexus therefore grow into the CNS along the concentration gradient towards these VEGF-producing cells (for a summary, see [77]). Besides the VEGF gradient, the ingrowth of blood vessels into the brain in a strictly radial manner might be supported by radial glial cells, which provide a preformed scaffold that the endothelial cells can follow [78]. A similar association of blood vessel growth guided by glial cells has been established for the vascularization of the retina [79] and could then also be traced to tracks formed by matrix-binding isoforms of VEGF [80, 81]. The question how a single growth factor could possibly accomplish functions like proliferation and migration of endothelial cells as well as vascular remodeling becomes more understandable when one considers that VEGF is expressed in several isoforms, which are produced by alternative splicing from a single gene. These isoforms differ by the absence or presence of domains that confer the ability to bind heparin and heparan sulfate proteoglycans in the extracellular matrix of cultured cells in vitro and therefore distribute differentially in the environment of VEGF-secreting cells in vivo [82]. Using mice either solely expressing a VEGF isoform lacking a heparin-binding domain or solely expressing a VEGF isoform binding to heparin, Ruhrberg and colleagues [83] elegantly demonstrated that differential diffusion rates and localization of different VEGF isoforms in the extracellular space in fact provide cues for regulating the vascular branch pattern during embryonic development, including brain angiogenesis. Although VEGF also seems to provide the most important angiogenic stimulus in the brain, it has been recognized that the precise “wiring” of the vascular system, i.e. establishment of the correct vascular network and patterning, does not occur without an ordered series of guidance decisions which are brought about by a number of guidance molecules initially discovered for axons in the


nervous system. In this context, the involvement of membrane-bound ligand receptor systems (such as the delta-notch, including the notch target genes Hey1 and Hey2 [84], the ephrinB-EphB families [85, 86], the robo-receptors and slit ligands [87], the netrins [88] and the semaphorins and their receptors of the plexin family [89]) has been demonstrated to play an important role in vascular development, including the brain. Additional growth factor-receptor systems have been implicated in the stabilization and maturation of the laid down and developing vascular network, including the CNS. In particular, Angiopoietin-1 and -2 (Ang-1, Ang-2) and their common receptor Tie-2 have been shown to be involved in vascular sprouting and remodeling, particularly in the adhesion to the ECM and in the recruitment of perivascular cells [90]. A common feature of Tie-2 and Ang-1 deficient embryos is a defect in the association of endothelial cells with the underlying extracellular matrix and with perivascular cells. Platelet-derived growth factor BB (PDGF-BB) has been shown to be actively involved in the vascularization of the brain by its relevance for pericyte recruitment, both in general and also including the BBB. Interestingly, mice deficient for PDGFBB show a dramatic decrease in pericyte investment of brain vessels, leading to the formation of lethal microaneurysm during late embryogenesis [91]. Additionally the TGFb pathway, ubiquitously important during embryogenesis, has been shown to have specific effects in angiogenesis through the receptors Alk-1, Alk-5 and endoglin and their intracellular effectors Smad5 and Smad6 (for a review, see [92]). Besides all these mechanisms, some evidence points to an involvement of the Wnt family of growth factors in vascular development. In canonical Wnt signaling, binding of the growth factor, which accumulates in the ECM [93, 94], to the frizzled receptor leads to the stabilization of cytoplasmic b-catenin, which involves proteins like disheveled and glycogene synthase kinase 3b (GSK-3b), leading to target gene transcription via a bipartite transcription factor formed by b-catenin and Lef/TCF [95–98]. In endothelial cells, little is known about the role of Wnt signaling during vasculogenesis or angiogenesis. However, the endothelial cell-specific deletion of the b-catenin gene was shown to lead to embryonic lethality around mid-gestation, due to vascular fragility, placenta and heart defects [99, 100], which at least for the heart defect could be linked to the transcriptional activity of b-catenin [99]. Regarding the development of the BBB, Wnt signaling might be of particular interest, as it has been shown that in particular brain endothelial cells are able to undergo canonical Wnt signaling. In vitro it could be demonstrated that primary mouse brain endothelial cells respond to Wnt-1 stimulation with activation of the canonical Wnt pathway involving b-catenin [101] and that endothelial cells growing into the neuroectoderm are positive for a Wnt signaling reporter, whereas other angiogenic vessels in the embryo are negative for Wnt signaling at all developmental stages investigated so far [102]. Support for a specific role of canonical Wnt signaling in brain vascularization comes also from some genetic diseases, like familial exudative vitroretinopathy (FEVR), in which the putative Wnt-receptor frizzled-4 (FZD4) is mutated, lead-

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ing to a lack of vascularization in the peripheral retina [103]. The FZD4 deficient mouse demonstrates a regression of vessels in the cerebellum, suggesting a role of the Wnt pathway for vascular maintenance in the CNS [104]. Although the exact role of Wnt signaling during brain vascularization remains to be elucidated, the results so far have to be seen in the light of previous reports, which claim that b-catenin protein expression is high during brain vascularization and becomes downregulated after BBB establishment [47]. Even though many of the molecular components involved in brain angiogenesis have now been identified, their exact mechanisms of action are still not fully understood. One major drawback for understanding brain angiogenesis is that these mechanisms also apply outside the CNS. Therefore, it seems unlikely that they are specifically involved in BBB differentiation to create highly specialized endothelial cells. However, the biggest remaining obstacle to the understanding of the roles of these proteins for the development of the vascular bed in the CNS is the fact that mutations in their genes invariably lead to lethal phenotypes during early embryogenesis before BBB differentiation starts.

1.5 Differentiation of the Blood-Brain Barrier 1.5.1 Permeability

When vascular sprouts enter the neural tube early during brain angiogenesis, vessels show a simple sinusoidal morphology and are characterized by their large diameter, irregular shape and the presence of diaphragmatic fenestrae. These vessels are permeable to small hydrophilic substances but probably not to macromolecules (see below). Along with their permeable phenotype, endothelial cells exhibit rudimentary tight junctions, indicated by substantial junctional clefts [105]. As development proceeds, the vessels lose their fenestrae, become smaller and thinner-walled and more regular in shape [54, 106]. In early fetal brain capillaries, the tight junction strands of endothelial cells are short and have low complexity, a pattern which changes dramatically during development, as the strands become longer and interconnected (i.e. complex) and the outer leaflets of adjacent membranes within junctional contacts seem to be fused in so called “membrane kisses” [19, 107]. As initially described, the P-face association of particles in freeze fracture analysis is another parameter to evaluate the tight junction maturity in mammals. Indeed, it could be demonstrated that, in general, the density of particles within tight junction strands increases late during embryonic development (after day 18 in the rat) and after birth a significant increase in the P-face association could be observed, representing a transition to the adult conformation of tight junctions [19]. An important controversy exists with regard to the development of the mammalian BBB [61, 108, 109]. Studies performed in analogy to the early studies of Ehr-

1.5 Differentiation of the Blood-Brain Barrier

lich, in which proteins or dyes were injected into the vascular system, showed that plasma proteins entered the fetal brain in significantly higher amounts than they entered adult brain [110–112] whereas others did not [113]. The former studies performed in the mouse and the chick even demonstrated that tightening of the barrier occurs as a gradual process, independent from vascular proliferation and starting late during embryogenesis when angiogenesis is not yet complete [114]. These studies were criticized based on the argument that, due to the small size of the fetus and its total blood volume, the amount of injected tracer as well as the injected volume were “enormous” and caused an artificial leakage of the developing BBB [108]. Based on immunohistochemical studies for native plasma proteins in fetal brains, which avoided the manipulation of small embryos and showed that there is very little plasma protein in fetal and newborn brain, it was argued that the barrier is present to plasma proteins as early as blood vessels form in the brain [115]. This controversy appears to be based on the lack of standardized quantitative BBB permeability measurements from the early fetal stage through later neonatal development to maturity, which might have simplified a comparison between the different species investigated. Comparison of BBB maturation between different species is extremely difficult due to the differences in their rates of brain development and especially since birth is not a reliable marker by which to stage BBB development [116]. Also, the independent maturation of the blood-cerebrospinal fluid barrier at the level of the choroid plexus epithelial cells is rarely considered in these measures [117]. In any case, endothelial barrier permeability to a small molecule, a-amino isobutyric acid (AIB), was shown to decrease in fetuses only late during gestation in sheep [116] and not until 17 days after birth in rabbits [118]. Additionally, permeability to ions as measured by electrical resistance of small pial blood vessels is several times higher in the fetus than in the adult and drops just before birth [119, 120]. Taken together, it seems that, depending on the species, small molecules access the fetal and newborn brain more readily than they access the adult. It should be stressed at this point, though, that BBB tightness is not just “switched on” at a specific time-point during brain angiogenesis but rather occurs regionally when angiogenesis is still ongoing and increases gradually [121].

1.5.2 Transport Systems and Markers

During BBB development, capillary brain endothelial cells acquire a characteristic set of transport systems, considered to be related to the development of the physical barrier (for detailed reviews, see [53, 57, 61, 77, 122–124]. The glucose transporter Glut-1, establishing a carrier-mediated transport of glucose, is one of the earliest BBB markers as it is already expressed in brain endothelial cells of the first vascular sprouts entering the developing neural tube. At that time in embryogenesis, neuroepithelial cells also express Glut-1 which is, however, rapidly downregulated whereas expression in brain endothe-

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lial cells increases [125]. At first, Glut-1 is equally distributed between the luminal and the abluminal membranes of endothelial cells [54]. As development proceeds, expression of Glut-1 on brain endothelial cells increases in response to the increasing demands of the developing brain for glucose, with higher localization at the abluminal versus the luminal membrane. Also, probably in response to the metabolic needs within the developing brain, the upregulation of the transferrin receptor, belonging to the receptormediated transporters, can be observed on endothelial cells forming a BBB but not on those within the choroid plexus or the CVOs [55]. Furthermore, the active efflux transporter P-glycoprotein becomes expressed in endothelial cells early during brain angiogenesis [126]. Expression of P-glycoprotein is required for the differentiation of the BBB [56] and seems to ensure the rapid removal of ingested toxic lipophilic metabolites from the neuroectoderm before the BBB has fully differentiated. Early expression of P-glycoportein at the BBB might be important to protect the developing brain from maternally ingested lipophilic molecules, since the placental barrier is ineffective against lipophilic molecules. It has been generally accepted that P-glycoprotein is localized to the luminal membrane of brain endothelial cells [127]. This viewpoint has, however, been challenged by the findings that, in human and primate brain, P-glycoprotein was immunolocalized to astrocytic endfeet rather than the luminal surface of cerebral endothelial cells [128]. It could well be that P-glycoprotein and other active efflux transporters are expressed in endothelial cells and pericytes and astrocytes working in concert to prevent entry of bloodborne lipophilic molecules into the brain parenchyme. The upregulation of other markers of the BBB, such as the nonreceptor tyrosine kinase lyn [129] and the Ig-superfamily member HT7 can be phenomenologically correlated to the development of BBB vessels, but their function in CNS endothelial cells still needs to be elucidated [130–132]. In contrast to the genes which become upregulated, the panendothelial cell antigen MECA-32 becomes specifically downregulated in mouse brain endothelia during BBB maturation [133]. As a consequence, MECA-32 antigen is absent on the mature cerebral endothelium, whereas it remains present on vessels outside of the CNS and the capillaries within the CVOs. Based on sequence comparison using the BLAST algorithm, the mouse MECA-32 antigen is the mouse orthologue of the rat PV-1 gene product and the human plasmalemma vesicleassociated protein. PV-1 was shown to be specifically localized to the diaphragms of fenestrated endothelia [134, 135]. This might explain the specific downregulation of the MECA-32 antigen during the maturation of the BBB, as brain vessels lose their fenestrations. Therefore, it seems unlikely that MECA-32 antigen is involved in the paracellular barrier function of brain endothelial cell involving adherens and tight junctions, but rather in the formation of diaphragmed fenestrations directly responsible for the efflux of bloodborne molecules. This seems to be true for brain and nonbrain endothelia lacking these type of fenestrations, also explaining why the vasculature of cardiac and skeletal muscle largely lacks MECA-32 in resting, noninflamed conditions [136].

1.5 Differentiation of the Blood-Brain Barrier

1.5.3 Extracellular Matrix

Agrin is a heparan sulfate proteoglycan that is required for the development of postsynaptic specializations at the neuromuscular junction. Absence of functional a-dystrophin in Duchenne muscular dystrophy (DMD) and in one of its animal models, the mdx mouse, leads to a reduction of a- and b-dystroglycan, members of a dystrophin-associated glycoprotein complex (DGC) in muscle, which bind to agrin but not to laminin [52]. As the mdx mouse displays a disturbed BBB due to a disorganized endothelial and astrocytic cytoskeleton, it is tempting to speculate that agrin is involved in the differentiation of BBB impermeability by establishing a symmetric polarization of endothelial cells and astrocytes. This is supported by the observation that, during chick and rat development, agrin accumulates in the brain microvascular basal lamina at the time when the BBB becomes less permeable to small tracers [137].

1.5.4 Putative Inductive Mechanisms

Although several aspects of BBB phenotypic development in brain capillary endothelial cells have been monitored, the crucial question concerning the induction of this differentiation process remains to be elucidated. It is obvious that, when brain angiogenesis starts, endothelial cells get in contact not only with various neuroectodermal cells like neuroblasts and with various glial cells and/ or their precursors [78], but also with mesodermal cells such as pericytes or microglial cells. That endothelial cells do not show a predetermination to the BBB phenotype was elegantly shown by chick-quail xenografts in which vessels of the coelimic cavity of the embryonic chick acquired BBB characteristics when growing into a developing transplanted quail brain [63, 138]. These observations provided direct evidence that the development of BBB characteristics in endothelial cells is not predetermined but rather induced by the microenvironment of the nervous system during embryogenesis. Due to the close apposition of astrocytic endfeet to the vessel wall, astrocytes and their precursors have immediately been implicated in BBB differentiation and/or maintenance (Fig. 1.2 b) [139, 140]. It was demonstrated, in vivo, that astrocytes are capable of inducing some BBB characteristics in vessels of the anterior eye chamber [141]. However, the importance of astrocytes in that particular experimental system was questioned by observations that iris vessels were impermeable to dyes, even in the absence of astrocytes [142]. It is widely accepted instead that, in vitro, astrocytes or conditioned medium derived from astrocytes induce BBB-like characteristic in endothelial cells [20, 143, 144]. For many years, little has been known about the nature of the astrocyte-derived signals promoting brain endothelial maturation, although a recent publication by Lee et al. [32] shed some light on the astrocyte-endothelial interaction.

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Fig. 1.2 a and b

Fig. 1.2 Three steps in BBB differentiation. (a) Brain angiogenesis: vascular sprouts radially invade the embryonic neuroectoderm towards a concentration gradient of VEGF-A, produced by neuroectodermal cells located in the ventricular layer. VEGF-A binds to its endothelial receptor, the receptor tyrosine kinase flk-1/KDR. The endothelial cellspecific receptor tyrosine kinase Tie-2 and its ligand Ang-1 are involved in angiogenic sprouting early during embryogenesis. The cerebral endothelial cells express Glut-1 and the MECA-32 antigen. The tight junctions (simple TJs) are permeable to small molecules. (b) Differentiation of the BBB: the phenotype of cerebral endothelial cells changes such that they lose expression of the MECA-32 antigen and start to express the HT7 antigen. Glut-1 is now enriched on the abluminal surface of the endothelium. De novo expression of P-glycoprotein and the nonreceptor tyrosine kinase lyn can be observed. The tight junctions become complex TJs, P-face-associated and thus also tight for small polar molecules. Phenotypic changes of endothelial cells are accompanied by their close contact with pericytes and astroglial cells. The molecular mechanisms involved in the interaction between pericytes and endothelial cells are partially characterized and have been shown to be important for vessel maturation within the CNS. Recruitment of pericytes along the differentiating BBB vessels is ensured by several mechanisms: (1) PDGF-B produced by endothelial cells

binds to its receptor PDGFR-b on pericytes, (2) N-cadherin enriched on the respective membranes facing the neighboring cell type interact with each other and (3) Ang-1 expressed by pericytes binds to the endothelial receptor tyrosine kinase Tie-2. Only recently, some light has been shed on the molecular interactions between endothelial cells and astroglial cells in the developing CNS: (a) endothelial cells produce LIF, which induces the maturation of astrocytes via the LIF-Rb and (b) due to the presence of vessels, the oxygen level increases and endothelial cells produce PDGF-B, both leading to an upregulation of SSeCKS in astrocytes. In turn, SSeCKS upregulates Ang-1 expression in astrocytes, which acts as an endothelial differentiation marker and positively influences the membrane localization of junction protein as ZO-1 and claudin-1. (c) The cellular elements of the mature BBB: despite the fact that the cerebral endothelial cells form the barrier proper, close contact with pericytes, astrocytes and maybe neuronal cells is required for the maintenance of the BBB. The presence of the heparan sulfate proteoglycan agrin in the endothelial basal membrane might be important for polarization of the cells involved in establishing the BBB. The precise molecular mechanisms involved in the supposedly paracrine crosstalk between the cellular elements required for BBB maintenance in the mature CNS remain unknown to date. Modified from an original figure in [170].

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Searching for astrocytic BBB maturation factors regulated by oxygen, the authors identified the potential tumor suppressor src-suppressed C-kinase substrate (SSeCKS, pronounced “essex”; human ortholog is gravin), which was upregulated upon increasing oxygen tension (see also Chapter 2). Overexpression of SSeCKS in astrocytes leads to a reduction of their VEGF expression in vitro. This observation correlates to in vivo observations where expression levels of SSeCKS are low in early embryonic stages when VEGF is high and they progressively increase during late embryogenesis and in postnatal stages, when VEGF is downregulated and angiogenesis finally stops. At the same time, SSeCKS was found to induce the expression of Ang-1 in astrocytes, a known pro-differentiation factor for endothelial cells, leading to an increased localization of ZO-1 and claudin-1 to cell-cell junctions in endothelial cells. Furthermore, SSeCKS can be induced in astrocytes by PDGF-BB [145], which in turn is produced by endothelial cells and also plays an important role in pericyte recruitment to blood vessels [146]. In this context, it is interesting to note that evidence has been provided that endothelial cells support the differentiation of astrocytes from their precursors. In particular, endothelial cells of angiogenic blood vessel in the brain express leukemia inhibitory factor (LIF), which specifically promotes astrocyte differentiation [147]. Taking these data together, one might imagine a scenario in which angiogenic endothelial cells in the brain induce first the differentiation of astrocytes through LIF and second the astrocytic expression of SSeCKS through PDGF-BB. In turn, astrocytes downregulate VEGF and upregulate Ang-1 production, ceasing endothelial angiogenesis and inducing barrier maturation, respectively (Fig. 1.2 b). Thus, astrocytes appear to play an important role in BBB differentiation, but it seems unlikely that they are sufficient to induce the complete spectrum of BBB properties in endothelial cells. This is supported by the observation that BBB characteristics in brain endothelial cells appear very early and prior to astrocyte differentiation and because astrocytes are present also in the CVOs, where capillaries do not form a barrier. Also, pial blood vessels, which are also considered to be part of the BBB, lack an association with astrocytic endfeet. Pericytes are a cell population found in close association with endothelial cells, even at very early stages of development and seem to be more prevalent on CNS capillaries than on other capillaries [17]. The function of pericytes in vivo was unclear for a long time [148], but recently it became evident that they are required for vessel maturation [91, 146, 149]. During brain angiogenesis several factors, such as Tie2/Ang-1 [150], PDGFR-b/PDGF-BB [91] and TGFb-1, as well as receptors and transcription factors of the TGFb signaling pathway, such as TGFb-receptor II, Alk-1, Alk-5, endoglin and Smad5, have been identified for proper endothelial-pericyte interaction in this process [151–156]. Furthermore, the adhesion molecule N-cadherin [157] has been demonstrated to be transiently expressed and to be required for the correct pericyte recruitment along the angiogenic capillaries in the developing chicken brain [157, 158]. At the endothelial pericytic interface, N-cadherin is exclusively linked to the actin cytoskeleton through b-catenin, which along with N-cadherin becomes downregulated during BBB maturation in the chicken [47]. Besides the role of b-catenin as a binding partner for N-cadherin,

1.6 Maintenance of the Blood-Brain Barrier

it is feasible that, in parallel, b-catenin is involved in transcriptional events as described above. Whether this coincides with Wnt signaling in endothelial cells and, at least to some extent, with the induction of the BBB phenotype is still unclear, but it is noteworthy that the known target genes of canonical Wnt signaling also include claudin-1, which is a tight junction protein possibly involved in endothelial barrier establishment [159]. Currently, the available information on Wnt signaling in ECs suggests that this pathway becomes activated in a very restrictive temporal and spatial manner. So far, the data obtained from knockout and reporter mice in vivo and from brain endothelial cells in vitro strongly favor a specific role of Wnt signaling in brain ECs, making it a good candidate pathway to be involved in BBB differentiation and maintenance. Last but not least, a possible involvement of neuronal precursor cells in inducing BBB characteristics in immature endothelial cells has also been suggested [160]. This notion is supported by the finding that aV-integrin deficient mice develop an abnormal vasculature and cerebral hemorrhages due to inappropriate association with brain parenchymal cells, such as neuroepithelial cells, glia and neuronal precursors, in the continued presence of normal endothelial cell/pericyte interactions [161]. The resemblance of the phenotype of b8-integrin deficient mice [162] to that of the aV-integrin deficient mice suggests that loss of aVb8 renders brain parenchymal cells incapable of normal adherence to the basal membrane of brain microvessels, which contains the aVb8-ligand vitronectin. These data point to the possible importance of the extracellular matrix for BBB differentiation either in mediating cell-cell contact or in storing soluble factors which act as inducers of BBB differentiation.

1.6 Maintenance of the Blood-Brain Barrier

Pathological conditions within the CNS like ischemia, inflammation or tumor growth, when the CNS microenvironment is changed, are typically accompanied by BBB dysfunction, emphasizing that the barrier characteristics in CNS endothelial cells are not simply “switched on” during embryonic development, but rather that continuing regulation of their maintenance, probably provided by the tissue microenvironment is necessary for a proper barrier function of endothelial cells in any circumstance. Eventually, leakiness of the BBB is also accompanied by the loss of the differentiated phenotype of BBB endothelial cells as supported by the re-expression of the MECA-32 antigen on brain endothelial cell in inflamed vessels surrounded by inflammatory cuffs [163] or the selective loss of immunodetectable tight junction molecules such as claudin-3 [29]. In the latter case, one might speculate that loss of claudin-3 under these pathological conditions could lead to a shift from P-face- to E-face-associated tight junctions that are therefore less tight. Furthermore, the extracellular matrix might contribute to BBB maintenance. In particular agrin, a heparan sulfate proteoglycan, was found in the basement

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membranes of vessels with special barrier properties like those in the brain; and agrin has been proposed to be involved in the development of the BBB, as described above [164]. Astrocytes have been shown to express agrin in vivo and in vitro; and therefore agrin could serve as a basal membrane factor maintaining BBB functions twofold, namely by maintaining the polarization of both the brain endothelium and also astrocytes in vivo. This polarization might be relevant for the appropriate spatial organization of transport systems in the cell membranes of endothelial cells and astrocytes working in concert to prohibit the entry of lipophilic metabolites endangering the appropriate function of neurons. In any case, it is noteworthy that the extracellular matrix of leaky blood vessels in malignant human brain tumors was found to be devoid of agrin [165]. Additional evidence for the necessity of continuous signals from the CNS microenvironment for brain endothelial cells to maintain barrier properties is provided by in vitro observations. When brain endothelial cells are isolated and cultured in vitro, they rapidly lose many of their BBB characteristics, including Pface-associated tight junctions, a permeability barrier, expression of specific transport system and, in contrast, acquire peripheral markers as the MECA-32 antigen, indicating that integrity of the BBB strictly depends on signals provided by the CNS microenvironment [166]. This has been elegantly supported by a recently established novel mouse in vitro model of the BBB consisting of the coculture of mouse brain endothelial cells with mouse glial cells, where a functional BBB is established and expression of the MECA-32 antigen is suppressed in brain endothelium as long as glial cells are present [167]. Taken together, not only the development but also the maintenance of the BBB is tightly regulated by the permanent interaction of endothelial cells with the CNS microenvironment.

1.7 Outlook

The vascular system in the brain develops in three phases: vasculogenesis, angiogenesis and barriergenesis [168]. During the past decade, there has been an explosion in our understanding of the molecules involved in vasculogenesis and angiogenesis. Although we know that inductive signals within the embryonic neural tube are responsible for committing the endothelium to barrier formation, inductive molecules and their receptors are still unidentified. Even the cellular sources providing the inductive signals, i.e. astrocytes, pericytes or neurons, remain a matter of debate. Differentiation of the BBB is a long and complex process involving a series of early and late events and gene expression in microvascular CNS endothelial cells might be highly dependent on the complex interplay of paracrine interactions with pericytes and astrocytic foot processes. The identification of factors instrumental for the induction of BBB characteristics in endothelial cells might

References

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Acknowledgments

I owe great thanks to Stefan Liebner for sharing his ideas on wnt signaling in the BBB and to Urban Deutsch for his critical discussion of this manuscript.

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2 Brain Angiogenesis and Barriergenesis Jeong Ae Park, Yoon Kyung Choi, Sae-Won Lee, and Kyu-Won Kim

2.1 Introduction

Brain vasculature begins with an initial network of pia vessels, called as the primary perivascular plexus that is derived from the differentiation of angioblasts [1, 2]. These brain vessels eventually differentiate into the complex microvessel network, which requires at least two distinct processes: brain angiogenesis and barriergenesis [2, 3] (see Chapter 1). Brain angiogenesis involves endothelial proliferation and migration, endothelial assembling, vessel branching, and sprouting from the preexisting vessels, while barriergenesis involves remodeling and maturation into the blood-brain barrier (BBB) properties [1, 3–8]. Therefore, brain angiogenesis and barriergenesis occur throughout the embryonic and postnatal stages of the developing brain. Intensive investigation into the development of brain vasculature has revealed the importance of signaling through the coordinated interaction with brain microenvironment. In the embryonic brain, active proliferation and migration of neuroglial progenitors from the subventricular zone (SVZ) to the cortex region result in the limiting of oxygen delivery by diffusion from the perivascular plexus. Subsequently, an oxygen gradient is generated and neuroglia-derived factors are released into the brain [8, 9]. The plexus may induce successive waves of angiogenic vessels toward the oxygen gradient and the factors. Thus, angiogenic vessels run between rows of migrating neuroglia, which can lead neuroglia to neuronal/astrocytic differentiation [10, 11]. Then, the new blood vessels will deliver oxygen and make contact with neuroglial progenitor cells. Accordingly, in the oxygenated brain, the endothelial cells (ECs) lining angiogenic vessels lie under the influence of the neural/glial environment, where they acquire a selective permeability barrier, the BBB [1, 3, 6, 12–15]. Therefore, elucidation of the cellular and molecular mechanisms of brain angiogenesis and barriergenesis in response to oxygen tension will provide an insight to delineate the developmental process of the BBB.


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2 Brain Angiogenesis and Barriergenesis

2.2 Brain Angiogenesis

Brain vessels are formed by angiogenic processes from primary perivascular plexus in the pia. In early embryonic brain, neuroglial cells from SVZ grow and migrates beyond the limit of oxygen diffusion from the perivascular plexus of the pia vessels [3]. This situation probably generates local hypoxia, which acts as a strong stimulus for the induction of angiogenesis. The generated hypoxia may trigger the growth of angiogenic vessels by hypoxia-inducible factor (HIF-1) [7, 16]. HIF-1 is a transcription factor that contributes to a variety of developmental and physiological events as well as several diseases [17, 18]. HIF-1 regulates a number of genes responsive to low cellular oxygen tension, especially angiogenesis. Examples include vascular endothelial growth factor (VEGF) and its receptors, platelet-derived growth factor-b (PDGF-b), basic fibroblast growth factor (bFGF), and erythropoietin (EPO) [19, 20].

2.2.1 Hypoxia-Regulated HIF-1 in the Development of the Brain

The existence of hypoxia in the developing brain has been found by using a hypoxia-specific marker, pimonidazole hydrochloride, which is converted by hypoxiaactivated nitroreductase into a reactive intermediate to form adducts covalently with cellular component in hypoxic regions [21, 22]. In the early embryonic stage, hypoxia is detected in the mesenchymal region and gradually spreads into neural tubes. Moreover, the hypoxic region is well matched to that of HIF-1a immunoreactivity. In embryonic (E) days E13 and E18 in mice, hypoxic regions seem to move to the olfactory lobe, some connective tissues of craniofacial regions, marginal layer, and the ventricular neuroepithelia of the cortex, where neuroglial cells proliferate and generate locally avascular regions [21]. At postnatal (P) day P3, hypoxic regions are spread throughout almost the whole cerebral cortex (Fig. 2.1). As angiogenic vessels run into all brain regions, hypoxic regions are likely to disappear and then rarely be detected in the cerebral cortex of P21, when brain angiogenesis ceases. In addition, it has been reported that mouse brain exposed to lower oxygen concentrations also accumulates HIF-1a [23, 24]. Based on above observations, the appearance of hypoxic regions spatiotemporally occurring during brain development seems consistent with the idea that neuroglial cells proliferate and migrate away from the existing blood vessels, creating an oxygen gradient in the brain tissue [21, 24]. The hypoxic regions stimulate the expression of HIF-1a to increase the oxygen supply through new blood vessel formation [25, 26]. Therefore, hypoxia is implicated as a critical factor for vascularization in the developing brain through HIF-1a activation. Further supporting the role of hypoxia in the developing brain, other lines of investigation have suggested that systemic inactivation of HIF-1a alters vascular as well as brain development. In HIF-1a–/– mice, abnormal neural development


Fig. 2.1 Immunohistochemical staining of hypoxic regions, VEGF, and occludin in developing rat brain. Positive immunoreactivity is represented by the brown color of the DAB. Scale bars: 100 lm. (Adapted from [49], with permission).

and a defect in cephalic angiogenesis have been observed [22, 27]. More direct support for a hypoxic role has been investigated through the study of conditional knockout mice, with specifically targeted HIF-1a genes in the neural precursor cells of the developing brain [28]. Neural cell-specific ablations of HIF1a, although ECs express HIF-1a, exhibit not only a reduction in neural cells but also a vascular regression. The defective vessels in the mutant are likely due to the disturbance of the hypoxic response from HIF-1a deficient neural cells. Therefore, hypoxia occurring in the development of the brain is likely a strong angiogenic signal to induce the formation of an embryonic vascular network [16, 17]. Given the widespread HIF-1 expression in the developing brain that experienced an oxygen gradient, the understanding of HIF signaling pathways can provide a clue for the investigation of hypoxia-induced angiogenesis associated with brain development.

2.2.2 Hypoxia-Inducible Factor

HIF-1 is composed of two subunits, an oxygen-sensitive HIF-1a subunit and an oxygen-insensitive HIF-1b subunit [29]. HIF-1a is stable under hypoxia, whereas it is rapidly degraded under normoxia. Each subunit has the basic helix-loop-helix (bHLH) and the PER-ARNT-SIM (PAS) domain. The N-terminal of HIF-1a contains bHLH and PAS that are required for dimerization and DNA binding. The C-terminal regions are required for degradation and transactivation, which

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include the oxygen-dependent degradation (ODD) domain, two transactivation domains (N-TAD, C-TAD), and an inhibitory domain (ID) [17]. HIF-1 activity is unlikely to be regulated in the transcriptional level, because of the rapid degradation of HIF-1a in oxygenated cells [17–19]. Rather, HIF-1a is regulated by processes involving stability and subcellular localization [30–32]. From the viewpoint of the availability of oxygen, the regulatory mechanisms of HIF-1 have been extensively studied [19]. Recently, insights into the oxygen-regulatory function of HIF-1a have come from studies on posttranslational modifications, such as ubiquitination, hydroxylation, acetylation, and sumolylation. Under normoxic conditions, HIF-1a is subject to rapid degradation by the process of the pVHL-mediated ubiquitin protease pathway. The ODD domain within HIF-1a interacts with the pVHL that is part of an E3 ubiquitin ligase complex for HIF-1a polyubiquitination, letting HIF-1a be exposed to the pVHL-degradation pathway [31, 33, 34]. In detail, the association of HIF-1 with pVHL is triggered by HIF prolyl hydroxylase (PHD) that hydroxylates the two prolyl residues, Pro402 and Pro564, which are located within the ODD domain of HIF-1a [35– 39]. In addition, factor inhibiting HIF-1 (FIH-1) exerts hydroxylation of an asparagine residue (Asn803) located in the C-terminal transactivation domain (C-TAD) [40–42]. Thereafter, FIH-1 downregulates transcriptional activation by apparently interfering with the recruitment of coactivator p300. These hydroxylases are dioxygenases requiring oxygen and 2-oxoglutarate, which transfer one oxygen atom to prolyl and asparagyl residues, creating hydroxylated amino acid, and the other oxygen atom reacts with 2-oxoglutarate, generating succinate and CO2 [43]. The hydroxylase activity is likely to link oxygen availability. Hence, the posttranslational hydroxylation plays a key role in the direct regulation of HIF-1a stability. While hydroxylation takes center stage in the regulation of HIF-1a stability, acetylation seems to be another important modification. Acetylation is mediated by ARD1, which has been identified as a protein interacting with the HIF-1a ODD domain [44]. Interestingly, ARD1 acetylates Lys532 residue within the ODD domain. The acetylation of Lys532 increases the proteosomal degradation of HIF-1a, whereas the mutation of Lys532 decreases the interaction with VHL and stabilizes HIF-1a. It is intriguing that mouse N-terminal acetyltransferase 1 (mNAT-1) is expressed in the developing brain, which may combine with ARD1 to form a functional acetyltransferase, suggesting an important role in dividing neuroglia [45]. In addition, recent analysis of the yeast two-hybrid assay reveals that Tid-1L [a mouse homologue of Drosophila tumor suppressor l(2)tid] interacting with pVHL accelerates the binding between HIF-1a and pVHL, which leads to reduced HIF-1a protein levels. Under hypoxic condition, degradation of HIF-1a is prevented, allowing HIF1a to accumulate and be stabilized within the nucleus. Then, HIF-1a dimerizes with HIF-1b to become an active transcription factor. HIF-1a and HIF-1b dimers recognize HIF-responsive elements (HREs) within the promoter of hypoxia-responsive target genes. Recent reports on the stability and transcriptional activity of HIF-1 come from the small ubiquitin-related modifier (SUMO) study. The modification by SUMO-1, sumoylation, is known to regulate transcriptional


Fig. 2.2 Posttranslational modification of HIF-1a. Under normoxia, HIF-1a is subject to rapid degradation by the pVHL-mediated ubiquitin-proteasome pathway. Hydroxylation on Pro402 and Pro564 and acetylation on Lys532 within the ODD domain of HIF-1a potentiate the interaction with pVHL.

activities and the primary function of which prevents proteins from proteosomal degradation [46]. SUMO-1 and HIF-1a are concomitantly increased in hypoxic neurons and brain [47, 48]. Ectopic expression of SUMO-1 demonstrates that SUMO-1 increases HIF-1a stability; and coimmunoprecipitation and colocalization studies reveal the possibility of sumoylation regulating HIF-1a in the brain. It is noteworthy that lysine residues in the ODD domain of HIF-1a are likely to serve the change of HIF-1a stability in a different oxygen tension through the process of ubiquitination, acetylation, and sumoylation. Until now, however, it is unclear which lysine residue of the ODD domain is in charge of HIF-1a ubiquitination. An overall schematic diagram of a molecular mechanism of HIF-1a regulation is represented in Fig. 2.2. To answer more detailed questions on how HIF-1a is involved in the BBB development, further studies on coordinated interactions between posttranslational modifications, including acetylation/deacetylation, hydroxylation, sumoylation, and ubiquitination, for HIF-1 activity seem warranted in the brain.

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2.2.3 Hypoxia-Induced VEGF

Proliferation and migration of neuroglia from SVZ increase oxygen consumption; and restriction of oxygen diffusion from pia vessels results in the appearance of hypoxic regions [9]. The reduction in oxygen availability, hypoxia, is expected to activate HIF-1a and then increase VEGF, which is the most potent angiogenic factor. VEGF colocalization with the region of hypoxia has come from immunochemical studies in the developing brain. VEGF is expressed in the neural tube and mesenchymal cells in the folding head, which is the region where pimonidazole adducts are formed [21]. VEGF colocalization with hypoxic regions starts from around E13 to P3 and then gradually disappears (see Fig. 2.1) [49]. VEGF is known as a survival factor for ECs, both in vivo and in vitro, through the AKT signal pathway and is also a permeability factor [50–53]. In addition, VEGF stimulates several stages: migration, proliferation, and survival of EC during angiogenic processes. VEGF is composed of four different isoforms (VEGF206, VEGF189, VEGF165, VEGF121) by alternative exon splicing. VEGF165, the predominant isoform, binds to VEGFR1 and VEGFR2 and also interacts with neurophilins (NRPs) that induce an enhancement of VEGFR-mediated signaling. Since a heterogeneous VEGF-deficient embryo results in an abnormality in the formation of blood vessels, VEGF is considered as a critical factor involved in angiogenesis during embryogenic and adult stages [54, 55]. In fact, partial inhibition of VEGF by Creo-loxP-mediated, tissue-specific gene targeting shows impaired organ development. In addition, region-specific knockout mice of VEGF shows that the invasion and growth of blood vessels in the developing brain are severely impaired [56]. The distribution of VEGF and VEGF receptors has been found in ECs and surrounding tissues, respectively [3, 23, 56–59]. VEGF from the neuroglial progenitors of SVZ interacts with VEGFR on ECs; and their bindings lead to stimulate the ingrowth of capillaries from the perivascular plexus during corticogenesis. In the postnatal brain, VEGF expression has been suggested to localize almost exclusively to neuronal population of cells in the cortex [57, 58]. At the late postnatal stage, VEGF is expressed more evidently in the glial endfoot-invested feeder vessels that penetrate from the meninges. By the late postnatal stage, the localization of VEGF has switched for being predominantly neuron to glia. Therefore, early localization of VEGF is restricted to cortical neurons; and then glial cells elevate VEGF expression during later developmental stages [23, 58, 60]. Spatiotemporal expression of VEGF correlates with hypoxia-driven angiogenesis and maintenance of the cerebral cortical vasculature. Along with hypoxia-induced VEGF, several growth factors, including IGF-1, FGF, and TGFb, induce VEGF under a variety of pathophysiological situations [61, 62]. This observation is considered as a possibility for mutual interaction between oxygen tension and growth factors in the microenvironment of the developing brain. These communications between oxygen tension and growth fac-


tors might facilitate the induction of proper brain angiogenesis. Such a consideration, however, needs further work to explore how these factors could be regulated by the change in oxygen tension.

2.2.4 Other Neuroglia-Derived Angiogenic Factors

The contribution of several growth factors to angiogenesis has been the subject of several recent papers. Hypoxia-induced EPO gene expression is under the control of widespread oxygen-sensing mechanisms [63]. EPO is a main regulator for erythropoiesis and is known to be a glycoprotein that is produced mainly in adult kidney and fetal liver [64]. The production of EPO has also been found at comparable levels in the lung, testis, and brain. The abundant expression of EPO and its receptor in all brain regions attracts a growing interest. Along with EPO expression in the embryonic brain, EPO receptor deficient mice have revealed an increased apoptosis rate, suggesting a role of EPO for normal brain development [65]. EPO has also been found to be neuroprotective in the brain following exposure to a variety of insults. With regard to the endothelial functions of EPO, it has been demonstrated that EPO is a potent stimulus for postnatal neovascularization and endothelial progenitor cell mobilization. Even if more work is needed to define the role of EPO in angiogenesis, it appears that the increased expression of EPO and its receptor in blood vessels during cerebral ischemia contributes to new vessel growth in the hypoxic region [66, 67]. Fibroblast growth factors (FGFs) are potent stimulatory proteins that play significant roles in embryonic development as well as in adults [68]. The FGF/FGFR system is detected in capillary ECs. Stimulation of FGFR induces endothelial proliferation, migration, and differentiation through classic MAP kinase pathways in cultured cells and embryonic explants cultures. Sprouty, an intracellular protein that is expressed during the embryonic vasculature, may function as a negative regulator of FGF signaling. Nevertheless, no defective vascular phenotype has been reported, even in the disruption of FGF or FGFR [69]. However, tissue-specific transgenic mice of FGF showed an increase in vessel density. Moreover, the finding of a poorly branched vascular bed and avascular region in transgenic mice that overexpress a dominant negative FGFR has demonstrated that FGFs play a role in developmental angiogenesis, affecting branching morphogenesis. Therefore, FGFs are likely to promote the formation of larger and better organized blood vessels, even though the molecular event that governs the remodeling and branching of blood vessels remains to be elucidated. Recent studies suggest that the same or similar factors that are employed for neuronal patterning also play an important role within the vasculature [70, 71]. These dual effects of the factors seem to contribute in anatomically similar architectures between nerves and vessels that are both highly branched and parallel in their paths. Examples include: Eph/ephrin, Neuropilin/Semaphorin/Plexin, Slit/Robo, Netrin, and so on.

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The Eph/ephrin family is the largest family of RTK characterized. The Eph/ ephrin families are membrane-bound proteins that function as a receptor-ligand pair. Eph/ephrin has been known to regulate cell repulsion, adhesion, and migration, suggesting the regulatory function of axon guidance through chemorepulsion. In addition, vascular remodeling regulated by Eph/ephrin has been found in embryonic vasculature. Ephrin B2 is expressed in arterial ECs, whereas its receptor is reciprocally expressed in embryonic veins, providing the evidence for a distinction between arterial and venous endothelium. The expression of ephrin may be regulated by the Notch receptor, sonic hedgehog (Shh), and TGFb, all of which are involved in angiogenesis, suggesting reciprocal interaction in angiogenic processes. In addition, the role of Eph/ephrin in angiogenic remodeling of primitive capillary network structure to more complex vascular network has been documented through targeted disruption of either ephrin B2 or EphB4. In addition to endothelial expression, ephrin B2 is expressed in smooth muscle cells and pericytes surrounding vessels as development proceeds [72, 73]. Eph/ephrin has also emerged as a critical regulator in postnatal vascular remodeling and angiogenesis in retinopathy prematurity. Neuropilins (NRPs) are extracellular receptors for VEGF and semaphorin, and developmentally regulate both axon and vascular migration [70, 74]. NRP1 is identified as a VEGF receptor and simultaneously acts as a receptor for semaphorin families that is implicated in the neuronal guidance. Coexpressed with VEGFR2, NRP1 enhances the binding of VEGF to VEGFR2, by NRP1 forming a complex with VEGFR2. The role of NRP in the endothelial function has been found in studies of knockout mice that lacked the maturation and remodeling of the vascular network [75]. Likewise, emerging evidence shows that the slit/robo families, known as axonal guidance cues, are also involved in the blood vessel guidance [70, 76]. Netrin-1, a neuronal guidance factor, has been shown to act as an angiogenic factor [77]. Implication of Notch signaling in vascular development is suggested by genetic defects [78]. There are four Notch receptors and five ligands (Jagged-1, -2, Delta-1, -3, -4), which are membrane proteins. All the receptors and ligands have been expressed in the vascular system, including capillaries, vascular smooth muscles, and pericytes. Studies of endothelial function have shown that Notch signaling is negatively involved in angiogenesis by blocking b-integrated adhesion and sprouting. The b-integrin signaling is known to be necessary for VEGF-mediated endothelial sprouting. A further link has been found showing that VEGF induces the expression of Notch-1 and Delta-4. Even if many factors involved in neuronal patterning are now being identified, their roles in vasculature are still unclear. Considering the strong parallels between these two systems, it is likely that the factors are regulated in a complex spatiotemporal interaction to shape the blood vessel network.

2.3 Oxygenation in the Brain: Brain Barriergenesis


Soon after ECs form primitive blood vessels by angiogenesis, commencement of blood flow is likely to provide sufficient oxygen tension and vascularize brain tissue. Once oxygenation in the brain occurs in a sufficient quantity, ECs lining the vessels seem to be able to develop the barrier phenotype with more complex tight junctions (TJs), according to the requirement of the brain microenvironment [3, 6, 49, 79–83]. Therefore, the remodeling process of the primitive blood vessels, barriergenesis, builds the functional blood vessels with the BBB. TJs in the BBB are composed of transmembrane proteins, including occludin, claudin, junctional adhesion molecule, and VE-cadherin. In addition to transmembrane proteins, TJs also include several accessory proteins that are necessary for structural support: zonula occludens (ZO) proteins, 7H6, cingulin, and so forth. TJ assembly is seemingly regulated by interactions between multiple protein kinases, including PKC subtypes [84, 85]. In fact, TJ proteins are intimately associated with cytoskeletal architecture [86]. Besides complex TJs, BBB characteristics include several key features. The BBB structure expresses GLUT as a glucose transport, aquaporin as a water channel, and p-glycoprotein as an efflux transporter. All these proteins of the BBB regulate extremely low permeability, restriction of a free exchange of molecules by specific transport systems, regulation of homeostasis of the central nervous system (CNS), and prevention of the CNS from the extravascular environment [83]. More importantly, the BBB is enhanced by the presence of differentiated astrocytes and pericytes [6, 10, 12, 81, 82, 87]. These cells can reinforce barriergenesis through their direct interaction with ECs and/or secreted factors and finally lead to mature, functional blood vessels.

2.3.1 Cellular and Molecular Responses Following Brain Oxygenation

In earlier developmental stages, the primitive and discontinuous blood vessels invade the brain in response to a hypoxia- and neuroglia-induced factor gradient. Then, sufficient oxygen supplied by the invading vessels gives a great effect on the maturation of both neuroglia and ECs [5, 80, 88]. Several factors secreted from angiogenic ECs, including leukemia inhibitory factor-1 (LIF-1), have been shown to induce astrocyte differentiation [11]. In ECs, TJ proteins including ZO-1 and occludin increase in the brain tissue (Figs. 2.1 and 2.3). In addition, confocal microscopy studies in the adult and developing human brain demonstrate that occludin and claudin-5 are expressed and assembled at the junctional areas [89]. In contrast to occludin expression for TJ formation, hypoxic regions and VEGF expression are slightly detected in the embryonic stage, spread through the whole cerebral cortex at the postnatal stage, and then gradually disappear (Fig. 2.1). Besides, the significance of oxy-

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Fig. 2.3 Immunohistochemical staining of SSeCKS, VEGF, and ZO-1 in developing mouse brain. Immunoreactivities were quantified by analyzing each stained area relative to stained areas of adult brain (SSeCKS and ZO-1) or of P3 brain (VEGF). (Adapted from [79], with permission).

gen tension in several in vitro studies has suggested that posthypoxic reoxygenation in cerebral endothelial cells regulates their permeability and TJ proteins [90]. Thus, it seems that the oxygen gradient from hypoxia to oxygenation might regulate brain angiogenesis and BBB maturation during brain development.

2.3.2 Role of src-Suppressed C Kinase Substrate in the Induction of Barriergenesis

Given that ECs are surrounded by perivascular astrocytes, oxygen diffusion from blood vessels may induce oxygen-regulatory factors in the perivascular astrocytes. A very attractive approach to search for oxygen regulatory factors has been accomplished using a cDNA representative differential assay (RDA) in rat primary cultured astrocytes. As a result, the src-suppressed C kinase substrate (SSeCKS; rat homologue of human AKAP12) from astrocytes under posthypoxic reoxygenation has been identified as an oxygen regulatory gene [79]. SSeCKS is known to be a potential tumor suppressor and to function as a multivalent scaffolding protein for PKC, PKA, calmodulin, cyclins, and b-adrenergic receptors [91–93]. SSeCKS can control actin-based cytoskeletal architectures [94]. In fact, SSeCKS expression is relatively ubiquitous, with highest expression in the gonad, smooth and cardiac muscle, lung, brain, and heart [95]. In adult brain, the expression of SSeCKS has been detected in the cerebral and cerebellar white matter [96]. Interestingly, exposure of rat primary astrocytes to reoxygenation following hypoxia leads to increased SSeCKS expression, compared with hypoxic exposure, suggesting that SSeCKS is regulated by oxygen tension [79]. Oxygen-induced SSeCKS in perivascular astrocytes seems to influence the adjustment of ECs directly or indirectly. The indirect effect of SSeCKS


on ECs shows that treatment with conditioned media from SSeCKS-overexpressing astrocytes reduces the endothelial capability for migration and tube formation, suggesting an antiangiogenic activity. Antiangiogenic effects of SSeCKSmediated signals seem to be related to the report that SSeCKS is a potential inhibitor for metastatic tumors and controls cell cycle progression by regulating the expression and localization of cyclin D [93, 97]. In addition, SSeCKS-conditioned media from astrocytes not only increase the expression of TJ proteins, ZO-1, ZO-2, claudin-1, claudin-3, and occludin, but also decrease the permeability of human brain microvascular endothelial cells (HBMECs), suggesting that SSeCKS may play a key role in barriergenesis [79]. Confocal microscopic analysis demonstrates that SSeCKS-conditioned media from astrocytes enhance the linear distribution of ZO-1 at the HBMEC margin, where ECs contact. The linear distribution of ZO-1 is turned into a discontinuous and broken margin after downregulating SSeCKS expression. In developing brain tissue, SSeCKS filamentous staining in surrounding blood vessels stained with ZO-1 is taken to show the interaction between SSeCKS-expressing astrocytes and ZO-1-expressing ECs. This interaction is in line with the idea that reoxygenation during later stages of brain development increases SSeCKS in perivascular astrocytes, which in turn upregulates the expression of TJ proteins in neighboring ECs. Thus, SSeCKS is involved in stabilizing the BBB structure in the brain. The cerebral cortical expression of SSeCKS and TJ proteins progressively increases in late embryogenesis. In adult mice, these proteins are abundantly and stably expressed. In contrast, the expression of VEGF is high from E11 to P3, then gradually decreases and is almost undetected in adult brain (Fig. 2.3). In addition, SSeCKS is co-localized with GFAP; and SSeCKS-expressing astrocytes closely interact with ECs lining blood vessels. An additional report is that platelet-dependent growth factor-BB (PDGF-BB) potently modulates SSeCKS expression in cultured smooth muscle cells [98]. The report implies a possibility that SSeCKS expression might be regulated by PDGF-BB and then affect BBB maturation. Even if a detailed understanding on the role of SSeCKS for BBB formation and integrity is still a prerequisite, it is known that SSeCKS-mediated signals in astrocytes induce the maturation and stabilization of permeable vessels by inhibiting brain angiogenesis and enhancing the expression of TJ proteins. In addition, the constitutive expression of SSeCKS in the adult brain may provide a stabilizing signal for BBB integrity under physiological conditions.

2.3.3 Barriergenic Factors in Perivascular Astrocytes and Pericytes Following Brain Oxygenation

Considering the importance of the brain endothelial barrier, it is likely that the list of factors involved in the signaling cascade might be large. There are numerous factors associated with maturation of vessels. Angiopoietin-1 (ang-1)/ Tie2 seems partly to be responsible for the stabilization and maturation of ves-

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sels via interactions between the matrix, pericytes, and ECs [99, 100]. Ang-1/ Tie2 is observed in both adult and embryonic stages and is upregulated during vascular formation [101, 102]. Mice lacking or overexpressing ang-1 have revealed that ang-1 is responsible for recruiting and sustaining periendothelial support cells and for contributing to the formation of leakage-resistant blood vessels [103, 104]. In addition, Tie2 deficiency normally produces the initial phases of angiogenesis and blood vessel formation, including sprouting [105]. However, blood vessels throughout the embryo are absent from the formation of normal hierarchical networks, suggesting a role of ang-1/Tie2 during the stages of EC maturation by cell-cell interaction. In vitro studies show that exposure of cultured astrocytes to posthypoxic reoxygenation results in a higher expression and secretion of ang-1 and a lower expression of VEGF than does hypoxic exposure of astrocytes [49]. The secretion of ang-1 from perivascular astrocytes can bind its receptor, Tie-2, onto ECs. Direct exposure of cultured ECs to recombinant ang-1 protein not only markedly increases the expression of TJ proteins but also decreases its permeability (personal observations). Similarly, it has been documented that ang-1 opposes the permeability action of VEGF [106]. Moreover, ang-1 suppresses VEGF-induced vascular permeability in the skin and diabetic retinopathy [107]. Additionally, when BBB breakdown occurs, lesion and perilesional vessels decrease ang-1, supporting the leakage-resistant role of ang-1. Other lines of investigation have demonstrated that brain pericytes are important for the control of EC growth and migration and the integrity of microvascular capillaries [108]. Consistent with the role of ang-1 for vascular remodeling processes, vascular pericytes secrete ang-1 in response to oxygenation. The pericyte-derived ang-1 induces occludin expression in brain capillary ECs through Tie2 activation in vitro, which results in barriergenesis. Hence, it may be possible that ang-1 secreted from astrocytes and pericytes stabilizes ECs through an increase in TJ proteins (see Chapter 5). As a mechanism for SSeCKS-mediated barrier formation, it has been investigated whether ang-1 might be a barrier factor [79]. Overexpression of SSeCKS in rat primary astrocytes markedly reduces the expression of VEGF. In contrast, SSeCKS overexpression leads to an increased expression of ang-1, related to the maturation of preexisting vessels. Simultaneously, ang-1-neutralized, SSeCKSconditioned media not only markedly reduce the expression of TJ proteins but also strongly increase vessel permeability. Therefore, SSeCKS is likely to enhance the barrier function of ECs through ang-1 signaling. These findings indicate that SSeCKS is likely to, at least in part, trigger vessel maturation and stabilization during BBB development by upregulation of TJ proteins mediated by ang-1. Accumulated evidence shows that PDGF-b plays a critical role in the recruitment of pericytes to newly formed vessels [109]. It has been reported that PDGF-b is expressed by sprouting endothelium and pericyte/vSMC progenitors, suggesting a paracrine mode of interaction between the two cell types. Moreover, endothelium-specific PDGF-b knockout leads to vSMC/pericyte deficiency,

2.4 Perspectives

but no obvious effect on the vasculature. From these results, the endothelial PDGF-b signal controls pericyte recruitment to angiogenic maturation of vessels. Exposure of astrocytes to oxygenation following hypoxia leads thrombospondin-1 (TSP-1) to increase and sustain for a while [49]. Through this finding, it can be envisaged that the oxygenation signal in the brain microenvironment induces TSP-1. The increase in TSP-1, in concert with a decrease in VEGF, seems to promote a developmental switch from angiogenic to a differentiated, quiescent state of ECs. TSP-1 has been known to be a major activator of TGF-b1, which is a multifunctional cytokine expressed in ECs and mural cells and which promotes vessel maturation by stimulating extracellular matrix (ECM) production and by inducing the differentiation of mesenchymal cells to mural cells. Furthermore, it was recently reported that TGF-b1 upregulates the TJ and P-glycoprotein of brain microvascular ECs. Therefore, this suggests that TSP-1-dependent TGF-b1 in the brain may keep the BBB functioning. Besides, glia cellderived neurotropic growth factor (GDNF), in the TGF-b family, is likely to be involved in BBB maturation [110]. Adrenomedullin (AM) has been identified as a vasodilator peptide and a new member of the calcitonin gene-related peptide (CGRP) family. AM is a peptide hormone with multifunctional biological properties, participating in the regulation of vascular tone, inflammation, and other physiological events of the vasculature. Northern blot and radioimmunoassay have revealed the AM distribution in the central nervous system, many neurons, ECs, and perivascular glia cells. The endothelial expression of AM plays a role in the induction and maintenance of BBB properties [111]. Moreover, interleukin-6 and bFGF are also involved in the regulation of the BBB [112].

2.4 Perspectives

Overall, many reports support the idea that oxygen tension and neuroglia-derived factors contribute to brain angiogenesis and barriergenesis. For the whole process, although an exception may exist, a change in oxygen tension is likely to cooperate with a variety of neuroglia-derived factors released in the microenvironment of the developing brain. These combined effects may contribute to the structural and functional integrity of the vascular system (Fig. 2.4). Endothelial, glial, pericyte, and neuronal cells at the level of individual cells are sure to respond to a variety of signals derived from oxygen tension. Therefore, further studies are required to lineate how complex signals mutually interact with and affect the proliferation and differentiation of the cells. These molecular and cellbased researches will be crucial to understand the development and maintenance of the BBB [71].

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Fig. 2.4 A proposed model for brain angiogenesis and barriergenesis. In the earlier developmental stage, excessive proliferation of neuroglia in the brain results in the reduction of the local oxygen tension, termed hypoxia. Hypoxia-induced HIF-1a in the neuroglia induces angiogenic factors (e.g. VEGF). They lead to an invasion of primitive angiogenic vessels by binding their receptors (e.g. VEGFR2) and mutual interaction, termed angiogenesis. The angiogenic vessels provide neuroglia with oxygen. In turn, hypoxia-induced HIF-1a is downregulated by an oxygen-dependent pathway

through the ubiquitin-mediated proteosomal degradation. Simultaneously, the oxygenated perivascular astrocytes increase the expression of SSeCKS, which regulates the secretion of ang-1 in the perivascular astrocytes. The secreted ang-1 binds to the Tie2 receptor expressed in ECs lining the blood vessels. The ang-1/Tie2 signal pathway triggers the upregulation of TJ proteins and gradually induces the TJ formation. Thereafter, the blood vessels are differentiated into mature, functional units with BBB characteristics, termed barriergenesis.

References

Acknowledgment

This work was supported by the Creative Research Initiatives Program of the Ministry of Science and Technology of Korea.

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3 Microvascular Influences on Progenitor Cell Mobilization and Fate in the Adult Brain Christina Lilliehook and Steven A. Goldman

3.1 Introduction

Stem cell expansion and mitotic neurogenesis in the adult brain, in those regions and species in which it occurs, is delimited to discrete niches for neurogenesis and gliogenesis. These niches are typically in close proximity to foci of active endothelial cell turnover within the local capillary microvasculature. Both neurogenesis and angiogenesis within these niches appear regulated by vascular endothelial growth factor (VEGF), which has direct actions on resident stem and progenitor cells, and also triggers a cascade of paracrine effects that can mediate progenitor cell mobilization and recruitment. In particular, by expanding the local capillary bed, VEGF induces the generation of both neuronal and glial differentiation agents by the activated microvasculature. This process is dynamically modulated by ambient cues of gliovascular origin that include nitric oxide and purine nucleotides, which can act to regulate the mobilization of both subependymal and hippocampal neural progenitors. The close interactions between angiogenesis and both neurogenesis and gliogenesis effectively specify the molecular environment that defines local niches for cell genesis in the adult brain. 3.2 Angiogenic Foci Persist in the Adult Brain

The capillary microvasculature of the brain is created during development, as the perineural vascular network covering the brain generates capillary sprouts that migrate in a centripetal fashion, inwardly to the neuroepithelium (see Chapter 1). In the rat, intracerebral vascularization starts at day E11.5 and heralds a period of high endothelial cell proliferation, during which intracerebral branches penetrate the cerebral anlagen and then elongate and ramify within it. During this process, growing blood vessels penetrate the brain parenchyma at least in part in response to VEGF secreted from cells in the ventricular zone (Breier et al., 1992). Endothelial cell migration is thus concurrent with and in Blood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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the opposite direction to neuronal migration from the ventricular wall. This process is largely complete within 2 weeks after birth, after which brain endothelial division is sharply down-regulated (Plate, 1999). In most adult somatic tissues, endothelial cells then become relatively quiescent, characterized by slow turnover unless specifically activated. Indeed, the adult brain exhibits little endothelial cell turnover, unless activated in the setting of hypoxic ischemia, inflammation or tumor invasion. Nonetheless, several discrete foci of persistent angiogenesis have been identified in the adult brain (reviewed in Greenberg and Jin, 2005). Remarkably, these appear to correspond to regions of persistent progenitor cell turnover and neurogenesis (Louissaint et al., 2002; Palmer, 2002). These observations have led to intensive investigations of the mechanisms by which the gliovascular unit may regulate progenitor cell mobilization and fate, both in the germinative neuroepithelial layers of the adult subependyma and hippocampus, and among the dispersed progenitor cells of the adult brain parenchyma.

3.3 Neurotrophic Cytokines Can Be of Vascular Origin

During the earliest stages of development, endothelial cells may support stem cell expansion (Shen et al., 2004). More broadly speaking, microvascular endothelial cells can exert context-dependent effects on progenitor cells that can vary dynamically with the environmental cues to which they become subject. In both the developing and mature CNS, endothelial cells in fact secrete a multitude of potentially neurotrophic cytokines and matrix moieties (Weinstein, 2005), and as such, may provide a developmentally regulated palette of cues to neural stem cells as well as their daughter cells, both neuronal and glial (Rak et al., 1996). Among these signals is BDNF, which has been identified as a differentiation factor for cells generated from neural stem and progenitor cells. Leventhal et al. (1999) first reported that endothelial cytokines may exert strong differentiative effects on neural progenitor cells and that this effect was mediated through endothelial BDNF (Leventhal et al., 1999; Louissaint et al., 2002). When adult rat subependymal zone explants were grown in coculture with endothelial cells, substantially increased neuronal outgrowth and prolonged survival were observed. Neither effect was contact-dependent: analogous results were obtained both with endothelial cells grown on porous inserts in coculture, and with conditioned media from endothelial cells (Leventhal et al., 1999). Both protein and RNA analysis revealed that BDNF, alone among the known neurotrophins, was strongly expressed by human vascular endothelial cells derived from both brain and umbilical cord. BDNF secretion in these endothelial cultures exceeded 100 ng g–1 protein, surpassing the level required for neurotrophism in a variety of systems. In addition, blocking available BDNF in these cultures by adding soluble trkB-Fc abolished the trophic effects of endothelial culture (Leventhal et al., 1999). This study indicated the possibility of a link between the capillary micro-

3.4 Angiogenesis and Neurogenesis Are Linked in the Adult Avian Brain

vasculature and neural progenitor cell activation, by identifying endothelial cells as a possible robust source of BDNF. BDNF’s role in supporting neuronal recruitment has proven key to understanding the role of angiogenesis in providing a neurogenic niche. In the adult mammalian brain, BDNF strongly potentiates neuronal differentiation and survival of resident progenitor cells (Ahmed et al., 1995; Kirschenbaum and Goldman, 1995). Its infusion and overexpression have each been associated with increased neurogenesis, both in typically neurogenic regions such as the olfactory bulb, and in otherwise non-neurogenic regions, most notably the neostriatum (Benraiss et al., 2001; Pencea et al., 2001). In addition, BDNF deficient mice exhibit markedly diminished neuronal recruitment to sites of postnatal neurogenesis. Although many cell types of the adult brain express BDNF, including neurons and astrocytes, its high-level expression by endothelial cells and pericytes can establish the local microvasculature as a significant source for BDNF. The selective localization of BDNF-expressing vascular beds, coupled with its local matrix sequestration and binding by astrocytes expressing the signal-incompetent truncated form of trkB, may effectively provide reservoirs of BDNF that may permit a resumption of neurogenesis following local progenitor cell activation.

3.4 Angiogenesis and Neurogenesis Are Linked in the Adult Avian Brain

The lack of robust angiogenesis in most regions of the adult mammalian brain, coupled with the limited areas of ongoing neurogenesis, has necessarily limited studies of the interactions between angiogenesis and neurogenesis in adult mammals. Yet gonadal steroid-modulated tissues are prominent foci of modulated angiogenesis in adult mammals; and the brain is one such target for gonadal steroid. Indeed, steroid-receptive regions of the adult brain have been associated with both androgen- and estrogen-induced angiogenesis. On that basis, steroid-induced angiogenesis has been extensively studied in adult songbirds, whose brains are characterized by several regions of androgen- and estrogen-responsiveness, that regulate sexually dimorphic behaviors. The best studied of these regions is the vocal control nucleus HVC, a structurally plastic neostriatal locus that regulates the acquisition and execution of song in adult male songbirds and whose architecture is dramatically modifiable by gonadal steroid levels (Nottebohm, 1981). HVC is highly neurogenic and adds neurons throughout life in an androgen- and estrogen-dependent fashion (Goldman and Nottebohm, 1983; Hidalgo et al., 1995; Rasika et al., 1994). In adult female canaries that normally do not sing, testosterone administration stimulates both neuronal addition to HVC and the acquisition of male-like song. Over 20 years ago, it was noted that testosterone-mediated neuronal addition was preceded by a sharp burst of endothelial cell division in HVC, such that local endothelial cell division rose over 20-fold within 10 days of androgen administration to naïve females (Goldman and Nottebohm, 1983). Since this burst of mitotic angiogenesis

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directly preceded the recruitment of new neurons to the adult HVC, it suggested a potential causal link between these processes. Since endothelial cells had already been shown to be potent sources of BDNF (Leventhal et al., 1999) and since BDNF had been shown to have powerful trophic effects on neurons generated from resident stem cells (Ahmed et al., 1995; Kirschenbaum and Goldman, 1995; Pincus et al. 1998), these observations suggested that, in the bird brain, endothelial BDNF might serve as a link between newly induced vasculature and neuronal recruitment to the new vascular bed. This proved to be the case. Louissaint et al. (2002) demonstrated that testosterone induced both VEGF mRNA and protein in the adult HVC and that this led directly to the mitotic angiogenesis previously noted in response to androgens. These newly generated endothelial cells arose within the existing microvasculature, which expanded in terms of both mean luminal area and diameter, but not in capillary number (Louissaint et al., 2002). The gonadal steroidmediated induction of VEGF within HVC occurred concurrently with that of its receptor, VEGFR2, by HVC endothelial cells, allowing their mitogenic response to VEGF. The resultant activation and expansion of the local capillary bed proceeded as new vessels budded from existing capillaries; and their penetration and elongation was associated with MMP-mediated degradation of the extracellular matrix, and was followed by an angiopoietin 1-associated recruitment of smooth muscle pericytes to stabilize the nascent vessels (Lilliehook, Louissaint and Goldman, unpublished data). These events preceded the local production of BDNF, which became robust after an initial delay of 1–2 weeks following the initiation of testosterone treatment. In situ hybridization revealed that this testosterone-induced increment in HVC BDNF was localized almost entirely to the newly expanded microvasculature. The dominant participants in this process appeared to be the endothelial cells themselves. In culture, purified HVC endothelial cells secreted modest levels of BDNF, which rose rapidly and more than 3-fold in response to testosterone (Louissaint et al., 2002). The importance of BDNF in permitting neuronal recruitment in the adult HVC had been established by Nottebohm and colleagues, who noted that BDNF infusions into the adult female HVC potentiated local neuronal addition (Rasika et al., 1999), similar to observations in the rodent brain that BDNF infusion or over-expression were sufficient to mediate neuronal recruitment from neurogenic regions of the ventricular zone (Benraiss et al., 2001; Pencea et al., 2001). Together, these data indicated that endothelial cells were a major source of BDNF in the adult brain, and very likely its major hormonally regulated source. Thus, hormone-activated angiogenesis induced the endothelial release of a potent neurotrophic cytokine, BDNF. These data suggested that the testosterone-induced, VEGF-mediated, angiogenesis-associated increment in endothelial BDNF was necessary for the recruitment of new neurons from endogenous progenitor cells in the adult HVC. A corollary of this postulate was that the blockade of angiogenesis might be sufficient to abrogate androgen-associated neurogenesis in the adult HVC. This proved to be the case, in that when androgen-stimulated angiogenesis was

3.5 Angiogenesis-Neurogenesis Interactions in the Adult Mammalian Brain

blocked using a pharmacological inhibitor of the VEGFR-2 tyrosine kinase, neurogenesis and angiogenesis were both suppressed (Louissaint et al., 2002). Thus, niches for cell genesis in the adult brain may be both spatially associated with and causally dependent upon foci of angiogenesis, the hormonal modulation of which may provide a means of normally regulating cell addition to the adult CNS. By comprising a potent physiologically regulated source of BDNF, brain microvascular endothelial cells may serve to direct the addition of new neurons to sites of capillary activation and expansion in the adult brain. Together, these observations causally linked angiogenesis to neuronal addition in the adult brain (Palmer, 2002).

3.5 Angiogenesis-Neurogenesis Interactions in the Adult Mammalian Brain

The interactions between angiogenesis and neurogenesis in the adult mammalian brain are in some ways less understood than their avian counterparts; and they may also be fundamentally distinct mechanistically. In most of the adult brain, endothelial cell division is infrequent, consistent with a marked suppression of angiogenesis after development (Engerman et al., 1967). Yet in the subgranular zone (SGZ) of the hippocampus, a persistently neurogenic region, endothelial cells continue to divide at a high rate (Palmer et al., 2000). In the SGZ, dividing cells are grouped into clusters of 5–20 cells, which typically lie adjacent to capillaries. Among BrdU-incorporating cells within these clusters, over a third express endothelial antigens. This suggests that angiogenesis and neurogenesis are both temporally concurrent and geographically coincident, and that this coincidence represents a defining feature of the local niche for neurogenesis in the adult brain. Indeed, whereas the interaction of neurogenesis and angiogenesis in the adult HVC is at a distance, and is mediated through paracrine intermediaries, proliferating neuroblasts and angioblasts in the mammalian hippocampus are contiguous, and apparently coactivated in common. VEGF is a principal candidate for coactivating angiogenesis and neurogenesis in the adult hippocampus. VEGFR-2/Flk-1, the high-affinity receptor for VEGF, is expressed by both BrdU-incorporating progenitor cells and by local endothelial cells, while its ligand, VEGF, is expressed within the same region (Palmer et al., 2000). As a result, locally active VEGF might directly signal to both proliferating angioblasts and neuroblasts. Indeed, local hypoxia created by local metabolic demand from newly generated neurons may effectively drive the process in a feedforward fashion, by sustaining HIF1a-mediated VEGF production and release. In addition to VEGF, IGF1 has been reported to drive both angiogenesis and neurogenesis in a variety of systems, including the adult hippocampus, and may thus act as another coactivator linking the regulation of angiogenesis and neurogenesis. It would seem likely that these agents act in concert, in that each has been reported to potentiate the other’s signal competence in a variety of systems, including angiogenesis. Nonetheless, the proximal stimulus for both

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IGF1 and VEGF production in the hippocampus is unknown – in contrast to the songbird HVC, where gonadal steroids induce both IGF1 and VEGF (Jiang et al., 1998; Louissaint et al., 2002). These observations indicate that, in the adult mammalian brain, as well as in its avian counterpart, neural progenitor mobilization may be dynamically regulated by the gliovascular unit. Yet besides these classic growth factor-signaled pathways linking angiogenesis and neurogenesis, several more primitive pathways appear to permit the vascular regulation of progenitor cell turnover and daughter cell fate. Among these, purinergic and nitric oxide signaling, two distinct but interacting pathways for rapid communication among contiguous cells, seem especially important in regulating the mobilization and fate of tissue progenitor cells. Both are intimately associated with and regulated by the local microvasculature, and as such can provide the means by which local microvascular cells can activate local progenitor cells.

3.6 Purinergic Signaling to Neural Progenitors Cells: the Gliovascular Unit as a Functional Entity

Purine nucleotides can act as extracellular transmitters, and have been implicated in interactions between neurons and glia, as well as between neural, blood, and inflammatory cells (Burnstock, 2002 b). Purinergic signaling has been implicated in the proliferation of a variety of undifferentiated cell phenotypes (Burnstock, 2002 a, b; Sanches et al., 2002; Wang et al., 1992). In the normal brain, ATP is released by astrocytes, for which it serves as an active signaling moiety and is responsible for the propagation of intercellular calcium waves among syncytially coupled astrocytes (Arcuino et al., 2002; Cotrina et al., 1998, 2000; Nedergaard et al., 2003). Acting through P2Y receptors, ATP can promote the proliferation of radial cells and astrocytes (Weissman et al., 2004), as well as brain microvascular cells (Burnstock 2002 b; Wang et al., 1992). Moreover, ATP has been identified as a mitogen for v-myc immortalized neural stem cells (Ryu et al., 2003). Purines can signal through the Ras/Raf/MEK/MAPK pathway (Tu et al., 2000), indicating the potential for significant cross-talk with FGF, EGF/ TGFa, PDGF, and erbB-driven neural mitogenic pathways, among others. These observations would suggest the possibility that astrocytic purines, and ATP in particular, might participate in the control of neurogenesis, suggesting a means by which glial activity might regulate neural progenitor cell mobilization. This might provide a basis for several studies that have reported both subventricular zone and hippocampal astrocytes as uniquely enabling neurogenesis by resident progenitor cells (Lim and Alvarez-Buylla, 1999; Song et al., 2002). ATP is a short-acting molecule, both temporally and spatially, in that it is rapidly degraded by extracellular ectonucleotidases. Two ectonucleotidases, nucleotide triphosphate diphosphohydrolases 1 and 2 (NTDPases 1, 2), have been reported to modulate the local processing and clearance of ATP so as to modulate

3.7 Nitric Oxide is a Local Modulator of Progenitor Cell Mobilization

the local levels of ATP to ADP, the most potent ligands for P2Y receptor activation, and their metabolites, AMP and adenosine. NTDPase-1 catalyzes ATP hydrolysis to ADP and AMP, and ADP’s to adenosine; it is selectively expressed in the brain by endothelial and smooth muscle cells (Braun et al., 2000), though ubiquitously so. In contrast, NTDPase 2 strongly localizes to areas of subventricular and hippocampal neurogenesis in the adult brain, within which it colocalizes with astrocytes (Braun et al., 2003). By this means, astrocytes expressing relatively high levels of ATP in regions of stem cell expansion may utilize NTDPase-2 to locally generate ADP, which may drive local neural stem cell expansion and neurogenesis. These cells may in effect partner with adjacent microvascular pericytes to then use NTPase-1 to rapidly clear the bioactive ATP and ADP, thereby delimiting these sites of stem cell expansion. Although it is unclear whether purine receptor activation selectively favors the generation of any one phenotype, it is important to note the colocalization of these foci of purinergic signaling with areas of expression of noggin (Lim et al., 2000), a potent inhibitor of the pro-gliogenic bone morphogenetic proteins (Mabie et al., 1997). Persistent noggin expression in the subependyma (Lim et al., 2000) and hippocampus (Chmielnicki et al., 2004) appears to inhibit daughter cells from BMP-dependent gliogenesis, permitting their maintenance as undifferentiated progenitors, and hence accessibility to neurotrophic differentiation agents such as BDNF (Chmielnicki et al., 2004). These observations suggest that multiple influences may converge in niches for cell genesis, to specifically permit neurogenesis. Angiogenesis, local restrictions on gliogenesis, and microvascular neurotrophic influences, such as BDNF and IGF1, might then be viewed as necessary concomitants for biasing new daughter cells to neurogenesis. Indeed, such permuted interactions may essentially define the locations of adult neurogenic niches, by specifying the distributions of ongoing progenitor cell mobilization and neurogenesis (Goldman, 2003).

3.7 Nitric Oxide is a Local Modulator of Progenitor Cell Mobilization

The net effects of peptidergic cytokines and purine signals, among others, on neural stem and progenitor cells depend upon the concurrent actions of other local cues. Of particular interest in this regard are recent observations that nitric oxide (NO), as released by both endothelial cells and neurons, is a powerful regulator of mitotic neurogenesis (Cheng et al., 2003; Packer et al., 2003). In normal brain, NO appears to be a negative regulator of neurogenesis, such that NO synthase inhibitors promote the rapid expansion of progenitor cells of both the olfactory subependyma and the hippocampal subgranular zone (Packer et al., 2003; Sun et al., 2005). Specifically, NOS inhibition may be sufficient to release NO’s tonic suppression of progenitor cell turnover, thereby allowing the rapid reentry of these otherwise quiescent cells into the cell cycle. Conceivably, the resultant rapid mobilization of parenchymal progenitor cells might yield different

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daughter phenotypes in a context-dependent fashion, as newly generated daughters respond to regionally specified cues. By this logic, since BDNF is a powerful trigger for neuronal differentiation, those cells mobilized by NOS suppression, that then encounter BDNF might be expected to differentiate as neurons. This is precisely what is observed in the striatal and olfactory subependyma, as well as in the hippocampus, in which nNOS inhibition is associated with increased mitotic neurogenesis. In contrast, since BDNF-associated neuronal differentiation may be modulated by NO signaling (Chen et al., 2005; Cheng et al., 2003), NOS inhibition might also serve to impede BDNF’s potentiation of terminal differentiation (Cheng et al., 2003). Nonetheless, mice deficient in neuronal NOS exhibited markedly elevated mitotic neurogenesis within both the olfactory subependyma and hippocampus, again pointing to the net suppressive actions of NO on progenitor cell mobilization in the adult brain (Packer et al., 2003). Interestingly, a recent study has come to different conclusions with respect to the normal and pathological roles of eNOS signaling, in that mice deficient in eNOS were noted to exhibit diminished subependymal and hippocampal neurogenesis, and correspondingly deficient compensatory neurogenesis following ischemia, relative to wild-type mice (Chen et al., 2005). These observations suggest that eNOS and nNOS may have very different effects in the regulation of progenitor cell mobilization. Since they generate the same product, the differences noted between nNOS and eNOS inactivation may be due to the different paracrine pathways influenced by NO of endothelial and neuronal origin, and to the distinct spatial and temporal patterns of signal integration experienced by progenitor cells in eNOS- and nNOS-deficient environments.

3.8 Parenchymal Neural Progenitor Cells May Reside Among Microvascular Pericytes

Besides the neural stem and progenitor cells of the ventricular zone and subgranular layer, abundant glial progenitor cells pervade both the gray and white matter of the adult brain. Yet despite abundant studies of their distribution and lineage potential, the anatomic relationships of glial progenitor cells in the adult brain parenchyma remains unclear. Scolding et al. (1998, 1999) identified OPCs in the adult human white matter on the basis of both A2B5 and PDGFa receptor expression; and each has been quantified as representing roughly 3% of all white matter cells. Nunes et al. (2003) arrived at similar estimates, both in histological sections and upon A2B5 antigen-based FACS, as have others. Each described adult human OPCs as small and highly ramified, appearing similar to microglia, but none of these studies assessed the positions of these cells relative to the capillary microvasculature. Interestingly, these nominally glial progenitor cells of the adult brain express the NG2 chondroitin sulfate proteoglycan, by which they are operationally defined in rodents. Yet in humans, microglia (Pouly et al., 1999) and pericytes as well as OPCs can express NG2 (Ozerdam et al., 2001, 2002). Thus, given their antigenic and morphological similarities, it

3.9 The Role of the Vasculature in Post-Ischemic Mobilization of Progenitor Cells

seems likely that some cells previously characterized as ramified microglia are instead parenchymal glial progenitors and vice versa, and similarly, that some putative adventitial pericytes might have been microglia or glial progenitors. Conceivably, the perivascular NG2+ pool could then be composed of distinct populations of OPCs, microglia, and pericytes that adventitiously coexpress the same chondroitin sulfate proteoglycan. As such, some fraction of perivascular cells previously considered as smooth muscle pericytes might instead be resident progenitor cells; indeed, these morphologically and antigenically similar phenotypes might even be lineally related (Yamashima et al., 2004). The identity of perivascular NG2+ cells garnered particular interest following the observation that glial progenitor cells retain the possibility for neurogenesis, once removed from the tissue environment (Belachew et al., 2003; Kondo and Raff, 2000; Nunes et al., 2003). As such, the parenchymal glial progenitor cell pool may represent a reservoir of potential new neurons; and, to the extent that these cells might be perivascular, they may comprise an especially responsive pool of potentially neurogenic parenchymal progenitor cells. In this regard, Belachew et al. (2003) reported the generation of new neurons from resident NG2+ progenitors of the hippocampus, specifically highlighting their generation from the tissue parenchyma. Okano and colleagues then reported the production of new granule neurons from the vascular adventitia of the adult monkey hippocampus, following transient ischemia (Yamashima et al., 2004). In the latter study, BrdU+ cells were found in proliferative clusters within the vascular adventitia, within which a third of the cells expressed the neural progenitor marker musashi 1. Following transient ischemia, not only did the BrdU-labeling index rise, but so did the amount of BDNF expressed by SGZ pericytes, which appeared to potentiate neuronal differentiation by the newly generated daughter cells (Yamashima et al., 2004). Together, these studies suggested that at least some fraction of parenchymal neural progenitor cells may be associated with the vascular walls, within which they may be identified or misidentified as NG2+ perivascular cells, and that in some settings, these cells may be recruited not only for gliogenesis, but also for local neurogenesis. However, these studies have also highlighted the need for rigorous distinction between parenchymal glial progenitor cells, pericytes, and microglia.

3.9 The Role of the Vasculature in Post-Ischemic Mobilization of Progenitor Cells

Compensatory replacement of striatal neurons from resident progenitors was identified in experimental stroke models by several groups, who described neuronal recruitment into the neostriatum after focal ischemic injury (Arvidsson et al., 2002; Jin et al., 2003; Parent et al., 2002). Similarly, Nakafuku and coworkers described compensatory replacement of hippocampal pyramidal neurons – which, despite their apparent dissimilarity from striatal medium spiny cells,

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comprise another periventricular subcortical neuronal pool (Nakatomi et al., 2002). In each case, the trigger for the mobilization and striatal migration of resident progenitor cells appears to have been local hypoxic ischemia, possibly mediated by post-ischemic increases in VEGF, BDNF, and other neurotrophic and angiogenic cytokines (Sun et al., 2003), coupled with the parenchymal migration of the newly generated cells along the adventitial surfaces of local capillaries. Indeed, it seems likely that the response of the brain’s persistent germinal layers, the subventricular and subgranular zones, to local ischemia may be to recapitulate and expand the local microvascular niches for cell genesis, differentiation, and migration. By this means, the microvascular contributions to progenitor cell maintenance and mobilization in the adult brain may be seen as important not only to the brain’s normal homeostasis, but as critical components of its response to injury – microvascular influences might essentially establish the gain by which the extent and nature of compensatory neurogenesis and gliogenesis are regulated.

Acknowledgments

Work mentioned in the Goldman Laboratory is funded by NIH grants R01NS29813, R01NS33106, R01NS52534, and R01NS39559, and by the National Multiple Sclerosis Society.

References

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Part II The Cells of the Blood-Brain Interface


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4 The Endothelial Frontier Hartwig Wolburg

4.1 Introduction

The original finding of Paul Ehrlich [1] that an infused dye did not stain brain tissue, together with the complementary observation of his pupil Ernst Goldmann that the very same dye, if applied into the cerebrospinal fluid, did stain brain tissue, led to the concept of a biological barrier between blood and brain (Fig. 4.1). Due to the free access of dye from brain ventricle to brain tissue, it was concluded that there is no cerebrospinal fluid-brain barrier. However, the staining of circumventricular organs and the choroid plexus in Goldmann’s experiment applying the dye into the general circulation (Goldmann experiment I) and the avoidance of staining of these organs in the experiment applying the dye into the cerebrospinal fluid (Goldmann experiment II) suggested the existence of a barrier between the cerebrospinal fluid and the blood (Fig. 4.1). The cellular basis of the barrier was unclear for decades. Today, we know that in most vertebrates the barrier is located within the endothelium (endothelial blood-brain barrier (BBB) in Fig. 4.1; only in elasmobranchs, the BBB is located in astrocytes) and in the epithelial choroid plexus cells and the tanycytes of the circumventricular organs (glial blood-cerebrospinal fluid barrier (BCSFB) in Fig. 4.1). The structures essentially responsible for the restriction and control of the paracellular flux between both epithelial and endothelial cells was identified as tight junctions. Originally, these intercellular contacts were studied exclusively by means of morphological methods. From the middle of the 1980s onward, the molecular organization of the tight junctions was unraveled step by step, but primarily in epithelial cells. Today, we overlook a multitude of molecules concerned with both the formation and the regulation of barrier properties, but we are far from understanding the molecular network which establishes the transcellular barrier. Endothelial cells are seemingly more complicated regarding barrier regulation than epithelial cells, because epithelial cells, but not endothelial cells, develop barrier properties in vitro (thus after isolation from their microenvironment) which are close at their physiological level in vivo. We have to take into account that the endothelial tight juncBlood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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Fig. 4.1 Topological scheme of the BBB. Gray lines mark the basal laminae (glia limitans superficialis et perivascularis). A = Astrocytes, BBB = blood-brain barrier, BCSFB = blood-cerebrospinal fluid barrier, CSF = cerebrospinal fluid, E = endothelium, GJ = gap junction, P = pericyte, PEn = choroid plexus endothelial cell, PEp = choroids plexus epithelial cell, S = synapses, TJ = tight junctions. Crossed or uncrossed arrows mean impermeability or permeability across barriers, respectively.

tions are elements responsive to the brain microenvironment, including the surrounding basal lamina as well as the “second line of defense” which consists of pericytes, astrocytes, and microglia. In this chapter, we will focus on the structure and function of the brain microvascular endothelial cells and then, in the last paragraph, touch on the great importance of “peri-endothelial” factors for the establishment of the BBB.

4.2 The Brain Capillary Endothelial Cell

Mature BBB capillaries in the mammalian brain are mainly characterized by the small height of the endothelial cells (Figs. 4.2 and 4.3 A), the interendothelial tight junctions [2] (for recent reviews, see [3, 4]; Figs. 4.3 to 4.6), the small number of caveolae at the luminal surface of the cell [5], and the high number of endothelial mitochondria [6]. In addition, the subendothelial pericytes are completely surrounded by a basal lamina (Fig. 4.3 B), phagocytic perivascular cells, and astrocytic processes belonging to the set of elements directly adjacent to the cerebral vasculature [7, 8].


Fig. 4.2

Fig. 4.3

Fig. 4.2 Electron microscopical ultrathin section of the rat brain. CL = capillary lumen, lined by the endothelium, N = neuron, NP = neuropil. Bar: 5 lm. Fig. 4.3 (A) Ultrathin section of a mouse brain capillary. The arrows point to the subendothelial basal lamina separating the pericyte from both the endothelial cell and the astrocyte. A = Astrocytic endfeet, E = endothelial cell, L = lumen, P = pericyte. Bar: 1 lm. (B) Ultrathin section of a rat brain capillary lined by endothelial cells (E) interconnected by tight junctions (TJ). The arrow points to a caveola at the luminal surface of the endothelial cell. P Pericyte completely surrounded by basal laminae. Bar: 0.5 lm.

The microvascular endothelial cells are doubtless most important in the restriction of BBB-related permeability. During brain angiogenesis and differentiation of the BBB, specific molecules have to be expressed in brain endothelial cells [9, 10]. For example, specific expression of the non-receptor Src family tyrosine kinase lyn has been demonstrated early during brain angiogenesis [11]. Recently, it could be shown that lyn is activated by hypotonic stress and phosphorylates a tyrosine of the transient receptor potential channel family member TRPV4 as a tonicity sensor [12]. Another important endothelial gene product encodes P-glycoprotein [13], which is required for the differentiation of the BBB [14] and seems to ensure the rapid removal of toxic metabolites from the neuroectoderm before the BBB has fully differentiated [15]. In the developing chicken CNS, it has been shown that angiogenic vessels invading the neuroectoderm express N-cadherin between endothelial cells and pericytes. With the onset of barrier differentiation, N-cadherin labeling decreased, suggesting that transient N-cadherin expression in endothelial and perivascular cells may represent an

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Fig. 4.4

Fig. 4.5 Fig. 4.4 Ultrastructural investigation of the passage of wheat germ agglutinin-horseradish peroxidase across bovine brain capillary endothelial cells, 12 days after coculture with rat astrocytes and two consecutive days after discontinued (A) or continued (B) coculture. In (A), the loss of astrocytic mediators is reflected by the free penetration of the tracer through the intercellular cleft into the subendothelial space. In (B), the intercellular cleft is occluded by tight junctions, which do not allow penetration of the tracer [49]. Bars: 1 lm. Fig. 4.5 Freeze-fracture replicas of tight junctions. (A) MDCK II cells have tight junctions, nearly completely associated with the P-face (PF). The arrow points to tight junctional strands. Image produced in cooperation with Dr. G. Fricker (Heidelberg, Germany). (B) The endothelial cells of bovine brain capillary endothelial cells have a high ratio of PF-associated tight junctions, which is higher than that of all other endothelial cells outside the brain. The arrow points to tight junctional strands. L = Lumen. (C) Bovine brain capillary endothelial cells in culture show a loss of PFassociated tight junctional particles. Most particles are associated with the E-face (EF). Arrows point to tight junction ridges which are only poorly occupied with particles. Bars: 0.2 lm.


initial signal which may be involved in the commitment of early blood vessels to express BBB properties [16]. The early adhesion between endothelial cells and pericytes might be the result of the release of chemotactic factors by endothelial cells to induce the migration of pericytes towards the endothelial cell wall and subsequent maturation of the vessels by an increased production of extracellular matrix components elicited by the action of activated TGF-b and other proteins [17]. Amongst these, platelet-derived growth factor (PDGF)-B, a high affinity ligand for the receptor tyrosine kinase PDGF-Rb present on pericytes is produced by endothelial cells during development. PDGF-B has been shown to be involved in vascularization of the brain as disruption of the PDGF-B gene leads to pericyte loss, endothelial hyperplasia, and lethal microaneurysm formation during late embryogenesis [18]. The fine structural investigation of endothelial cells in these PDGF-B- and PDGF-Rb-deficient mice showed a malformation of brain endothelial cells characterized by the folding of the luminal surface [19]. Interestingly, this increase in the luminal surface is also a typical feature of the blood vessels in the pecten oculius, which is a convolute of vessels within the vitreous body of the avian eye [20]. In these vessels, the pericytes die by apoptosis during development [21]. Thus, both the physiological loss of pericytes in the pecten oculi and the pathological loss of pericytes in the PDGF-Rb-deficient mouse lead to a characteristic alteration of the shape of endothelial cells, suggesting a role for pericytes in the morphogenesis of microvessels. Establishing the barrier is accompanied by further changes in the phenotype of the brain endothelial cells, such as upregulation of the HT7-antigen/basigin [22–24] or downregulation of the MECA-32 antigen [25]; and the expression of specific transporters and metabolic pathways can also be observed [26]. Concerning the development of the barrier function of brain capillaries [10, 27–32] it has become likely that BBB tightness is not just “switched on” at a specific time-point during brain angiogenesis, but rather the tightening of the barrier occurs as a gradual process which is independent from vascular proliferation and begins late during embryogenesis, when angiogenesis is not complete [33]. However, it should be stressed that an unequivocal correlation between tight junction structure and measured permeability has not been shown so far [34, 35]. The molecular mechanisms involved in barrier maturation are poorly understood yet. From transplantation studies showing that vessels derived from the coelomic cavity gain BBB characteristics when growing into an ectopic brain transplant [36], it is known that the development of BBB characteristics in endothelial cells is not pre-determined but rather is induced by the neuroectodermal microenvironment.

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4.3 Endothelial Structures Regulating Transendothelial Permeability 4.3.1 Tight Junctions 4.3.1.1 Morphology of Tight Junctions In endothelial cells, tight junctions as specialized contact zones were already known from ultrathin sections (Figs. 4.3 B and 4.4) [2, 4, 5] and, in epithelial cells, their morphology was described in detail by Farquhar and Palade [37]. Around the same time when the endothelial nature of the BBB was detected by Reese and Karnovsky [38] and classically described in the comprehensive study of Brightman and Reese [2], a novel technique was developed which was suitable to visualize the morphology of cytoplasmic membranes and intercellular contacts and to corroborate the then novel fluid mosaic model of membranes [39]. This technique was the freeze-fracture technique, and soon after its inauguration, tight junctions were the issue of many-fold freeze-fracture descriptions (as summarized by [40] and, more recently, by [41]), including endothelial cell tight junctions of the BBB (Fig. 4.5) [42–48]. If sectioned transversally, the tight junction appears as a system of fusion (“kissing”) points, each of which represents a sectioned strand. These strands form the real obstacle proper within the intercellular pathway, which are under close control of the brain microenvironment, in particular the astrocytes (see Sect. 4.4 of this chapter and, e.g. [49]; Fig. 4.4). Two parameters can be visualized by freeze-fracture electron microscopy: the complexity of strands and the association of particles with the inner (P-face) or outer (E-face) lipidic leaflet of the membrane. The complexity of the tight junction network was recognized to be related to the transepithelial electrical resistance [50]. Epithelial tight junctions are mostly associated with the P-face, forming a network of strands and leaving grooves at the E-face which are occupied by only a few particles (Fig. 4.5 A) [51, 52]. After ATP depletion, MDCK cells suffer from deterioration of the paracellular barrier (“gate”) function, which is accompanied by a reorganization of the actin cytoskeleton [53, 54] and a decreased P-face association of the tight junctions. Thus, the degree of particle association to the P-face seems directly to correlate with the observed transepithelial resistance. The tight junction structure of the brain capillary endothelial cells was investigated by Nagy et al. [47], again using the freeze-fracture method. They found that the brain endothelial tight junctions were the most complex in the whole vasculature of the body. This was in nice correlation to the hypothesis of Claude [50], according to which there is a logarithmic relationship between the number of tight junction strands and the transcellular electrical resistance. However, as pointed out already for epithelial tight junctions, the association of the tight junction particles with the P-face or E-face of the membrane has been described to be a further parameter of the quality of the endothelial permeability barrier in the brain [48]. The BBB tight junctions are unique among all endothelial

4.3 Endothelial Structures Regulating Transendothelial Permeability

tight junctions in that their P-face association is as high as, or even slightly higher than their E-face association (Fig. 4.5 B). Interestingly, the P-face/E-face ratio of BBB tight junctions continuously increases during development [29]. In cell culture, the freeze-fracture morphology of BBB endothelial cells is similar to non-BBB endothelial cells (Fig. 4.5 C), indicating that the association of the strand particles with the membrane leaflets reflects the quality of the barrier and is under the control of the brain microenvironment.

4.3.1.2 Molecular Biology of Tight Junctions The molecular biology of tight junctions has become extremely complex in recent years. As claimed by us elsewhere [3, 55, 56], most data have been found for epithelial cells, probably because the regulation of BBB endothelial cell tight junctions is considerably more complex than that in epithelial cells. Generally, the molecular components identified at tight junctions can be separated into different classes based on their structures and functions. First, there are the integral membrane proteins occludin and the members of the claudin family, and, as detected more recently, Ig-superfamily members such as the junction-adhesion molecules of the JAM group and the endothelial cell-selective adhesion molecule ESAM (see below). Second, there are adaptor proteins which are distinguished according to their function to be first- or second-order adaptors. First-order adaptors are based on their direct association with the integral tight junction proteins via PDZ domains and include, for example, ZO-1, ZO-2, and ZO-3 (see below). Second-order adaptors are based on their indirect association with the integral tight junction proteins and include, for example, cingulin or the newly described cingulin-related JACOP (see below). Furthermore, there are other proteins involved in signaling cascades, such as G-proteins, regulators of G-protein signaling, and small GTPases which have been reviewed elsewhere [56, 67, 68]. In the past ten years, the knowledge of the molecular composition and regulation of the tight junctions has rapidly extended [3, 4, 57–69]. Occludin and the claudin family are the most important membranous components, both of which are proteins with four transmembrane domains and two extracellular loops (Fig. 4.6). Occludin Occludin was the first tight junctional transmembrane molecule discovered [57]. It was initially isolated from junction-enriched membrane fractions of chicken liver as a transmembranous tight junction protein of approx. 65 kDa, which exists in several isoforms. Occludin shows high interspecies variability between chicken and mammals [58], sharing less than 50% identity in amino acid sequence. In contrast, human, mouse and dog occludins are more closely related, showing approximately 90% identity. Beside the high content of tyrosine and glycine in the first extracellular loop (approx. 60%), the most conserved region of occludin comprises the carboxy terminal ZO-1 binding domain, an a-helical coiled-coil structure, putatively linking occludin to the cytoskeleton.

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Surprisingly, the tight junctions in occludin-deficient mice [70] were not affected morphologically; and transepithelial resistance as measured in small and large intestine epithelial cells was not altered, compared to wild-type mice. However, the mice developed chronic inflammation and hyperplasia of the gastric epithelium, calcifications in the brain and around brain vessels, thinning of bones, postnatal growth retardation, testicular atrophy, and abnormalities in sexual behavior [70]. The authors concluded that occludin might have a function in tight junction modulation via the induction of intracellular signaling. Moreover, occludin is not required for the formation of tight junction strands. In a number of reports, posttranslational modifications of occludin such as phosphorylation of its cytoplasmic domains or binding to a ubiquitin-ligase [71] have been described as part of tight junction regulation [72–76]. For example, DeMaio et al. [77] reported on a clear reduction of occludin content in cultured aortic endothelial cells by shear stress (100 lN cm–2), but a time-dependent increase of occludin phosphorylation which could be attenuated by dibutyryl cAMP. Also, an increase in occludin phosphorylation has been described after treatment with VEGF [78], suggesting that hormonal and mechanical changes are able to increase the paracellular permeability by an early increase in occludin phosphorylation and a subsequent decrease in the occludin content. Taken together, it seems that mature cells need occludin to regulate rather than establish their barrier properties. The Claudin Family The claudins are the tight junction molecules which seem to fulfill the task of establishing barrier properties [60, 69, 79, 80]. Claudins share the same overall organization as occludin with four transmembrane domains, but have no sequence homology to occludin. The first claudins identified were isolated from chicken liver junctional fractions and were called claudin-1 and claudin-2 [59]. Since then, a number of related proteins has been identified and at present the claudin family contains more than 20 members [81]. It is now believed that claudins are responsible for the regulation of paracellular permeability through the formation of homotypic and heterotypic paired strands (for reviews, see [62, 69]). In this model, ion selectivity is achieved through the selective expression and combination of distinct claudins in certain tissues [82]. Therefore, it is not surprising that the claudins are not randomly distributed throughout the organs, but at least in part show a tissue-specific expression pattern. For example, claudin-5 was originally described to be restricted to endothelial cells [83], although it was recently also found in surface cells of the stomach and the large and small intestine [84]. In addition, claudin-16 is selectively expressed in the thick ascending limb of Henle in the kidney, where it selectively regulates permeability for Mg2+ ions [85]. Functional investigations support the view that the composition of the claudin species directly determines barrier function [80]. Tight junction-negative L-fibroblasts when transfected with claudin-1 or claudin-3 form tight junctions, which appear in freeze-fracture replicas associated with the P-face [79]. When transfected with claudin-2 or claudin-5, the cells form tight junctions associated with


the E-face [59, 83]. In contrast, occludin was found to be localized at both fracture faces [86]. Whereas occludin induced the formation of short strands, the claudin-induced strands were very long and branched resembling endogenous tight junctions [79, 87]. Transfection of MDCK cells with claudin-1 increased the transepithelial resistance about 4-fold and reduced the paracellular flux [88]. Transfection with claudin-2 of high-resistance MDCK I cells that normally express claudin-1 and claudin–4 mimicked both the resistance behavior and the tight junction morphology of low-resistance MDCK II cells [80]. Claudin-4 was formerly known as the Clostridium perfringens enterotoxin receptor (CPE-R). By treatment of MDCK I cells, Clostridium perfringens enterotoxin (CPE) selectively removed claudin-4 from the tight junctions. Tight junctions were disintegrated to a simple network with only a few anastomosing strands and the transepithelial electrical resistance (TER) was decreased. After CPE removal, barrier properties were re-established [89]. These results suggest that the combination and stoichiometry of the claudins might be responsible for the outcome of a given resistance or permeability. The claudins detected in endothelial cells were initially claudin-1 and claudin5 [83, 90]. Later, claudin-3 and claudin-12 were also found to be expressed by BBB endothelial cells [91, 92]. Therefore, at least four claudins (claudin-1, -3, -5, -12) are expressed by BBB endothelial cells. However, claudin-1 seems to be present in BBB endothelial cells but not targeted to the tight junction [4, 91]. Concerning claudin-5, the deficiency of this molecule as reported by the Tsukita group [92] could be expected to compromise the quality of the BBB and therefore the viability of the animal. However, astonishingly, new-born knock-out animals did not differ from the wild type in terms of macroscopic morphology; and even electron microscopy analysis did not reveal alterations in tight junctions. However, since a low molecular weight tracer (e.g. Hoechst dye) and not the higher molecular weight tracer microperoxidase extravasated from the brain vessels of the claudin-5 knock-out mouse, it was concluded that the claudin-5-based tight junctions restrict permeability for small molecules, < 800 Da [92]. Leaky rat lung endothelial cells transfected with a mutated claudin-5 (substitution of Thr207 by Ala) reconstituted a paracellular barrier against inulin (5 kDa) and mannitol. However, phosphorylation of the wild-type claudin-5 at Thr207 by cAMP was responsible for a rapid decrease in transendothelial electrical resistance (TEER) and a loosening of the claudin-5-based barrier against mannitol but not inulin [93]. Also, transfection of MDCK cells with claudin-5 increased TEER and reduced the permeability to ions [94]. These data together with the results reported by Nitta et al. [92] could also suggest that other tight junction molecules such as claudin-3 or claudin-12 and their association with claudin-5 are responsible for establishing the paracellular barrier against low molecular weight molecules. As mentioned above, claudin-3 and claudin-5 if transfected into cultured fibroblasts associate with the P-face and the E-face, respectively [59, 89]. BBB endothelial cells in vivo reveal a P-face/E-face ratio of about 55%/45% [29]; and as claudin-3 and claudin-5 are well expressed, it can be suggested that the degree of association with one or the other leaflet roughly reflects the stoichiometry of

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claudin expression in the tight junctions. In non-BBB endothelial cells, tight junctions are almost completely associated with the E-face and claudin-3 is rarely or not expressed. BBB endothelial cells cultured in vitro develop tight junctions which are associated with the E-face [48] and express less claudin-1 [90]. However, an antibody was used which is now known to recognize claudin3 as well. Under pathological conditions such as malignant glioma or experimental allergic encephalomyelitis, claudin-1/-3 was found to be lost and/or the tight junctions were E-face-associated [91]. Immunoglobulin-Like Proteins at Tight Junctions Almost simultaneously with the identification of claudins, a junctional adhesion molecule (JAM) has been reported as the first member of the immunogloblin (Ig) superfamily to be present at tight junctions (Fig. 4.6) [95]. JAM, which is now called JAM-A, localizes at homotypic cell-cell contacts of endothelial and epithelial cells and is highly enriched at tight junctions [95, 96]. Two Ig-like proteins closely related to JAMA, JAM-B, and JAM-C have been identified recently [97, 98]. JAM-B and JAM-C are restricted to endothelial cells and are largely absent from epithelial cells. Although ultrastructural analyses for JAM-B and JAM-C in endothelial cells are still missing, the co-localization of JAM-C with occludin and the zonula occludens protein-1 (ZO-1; see below) upon ectopic expression in MDCK epithelial cells suggests its localization at tight junctions [98]. Additional evidence for a role of JAMs in the formation of tight junctions is based on the observation that anti-JAM-A antibodies as well as soluble JAM-A negatively affect the formation of functional tight junctions after Ca2+ switch-induced cell-cell contact formation [96, 99] and on the identification of cytosolic proteins which associate with JAMs and which are implicated in the formation/function of tight junctions (see below). In this respect, it is interesting to note that JAM-A and JAM-B are expressed by Sertoli cells in the testis where they could be involved in the formation and/or maintenance of the blood-testis barrier [100]. Another role of JAMs in endothelial cells might be related to their predicted function in regulating leukocyte-endothelial cell interaction during inflammation, through homophilic and heterophilic interactions (for recent reviews, see [101–103]). Originally, blocking JAM-A was found to inhibit leukocyte diapedesis in vitro and during inflammation in vivo [104]. However, there is a considerable body of evidence that leukocyte diapedesis does not follow the paracellular route, but the transcellular route via a mechanism called emperipolesis (for overviews of both the classic transmigration studies and the recent literature, see [105, 106] and also Chapter 24). The transcellular mechanism as observed in experimental allergic encephalomyelitis (EAE) leaves tight junctions intact and implies a complex rearrangement of the luminal and abluminal membranes [107]. This does not exclude the possibility that, despite leaving tight junctions morphologically intact during transcellular transmigration, these junctions can molecularly be changed. In EAE, we demonstrated a selective loss of anti-claudin-3 immunoreactivity [91]. This molecular alteration is associated with an increase in vascular permeability, but not an opening of tight junctions


for paracellular leukocyte diapedesis. If we therefore assume that transcellular migration of leukocytes delivers a signal to the endothelial junction – probably via the actin cytoskeleton – it is tempting to speculate that functional antibodies that “tickle” junctional molecules such as VE-cadherin, PECAM-1, the JAM family or CD99 might trigger these intracellular signaling cascades such that they increase or decrease the endothelial mechanisms required for either transcellular or paracellular migration. More recently, four additional Ig superfamily members have been identified at tight junctions. These include the coxsackie and adenovirus receptor (CAR) [108], endothelial cell-selective adhesion molecule (ESAM) [109], junctional adhesion molecule (JAM) 4 [110] and the coxsackie and adenovirus receptor-like membrane protein (CLMP) [111]. They share with JAM-A, JAM-B, and JAM-C a similar organization, with two Ig-like domains. However, they are more closely related to each other than to the three JAMs and thus form a subfamily within the tight junction-associated Ig superfamily members [103]. Interestingly, CAR, ESAM, and JAM4 end in a type I PDZ domain-binding motif, whereas JAM-A, JAM-B, and JAM-C end in a type II motif, which suggests functional differences between the two subfamilies. The function of these four Ig superfamily members at tight junctions is not clear. CAR, JAM-4, and CLMP are predominantly expressed by epithelial cells, whereas ESAM is expressed exclusively in endothelial cells, including those in brain capillaries [109, 112]. Endothelial cells derived from ESAM-deficient mice display defects in endothelial tube formation, suggesting a role for ESAM in endothelial cell contact formation [113]. How this function relates to its specific localization at tight junctions is not yet clear. Peripheral Membrane Components at Tight Junctions The transmembrane proteins associate in the cytoplasm with peripheral membrane components which form large protein complexes, the cytoplasmic “plaque”. ZO-1, a 220-kDa phosphoprotein, was the first peripheral membrane component identified and characterized at tight junctions [114]. In cellular systems with less elaborate or no tight junctions at all, ZO-1 is found enriched in regions of the adherens junctions [115], where it may interact with components of the cadherin-catenin system [116, 117]. Since the discovery of ZO-1, many further components of peripheral tight junction proteins have been described (Fig. 4.6). One type of plaque protein consists of adaptors, proteins with multiple protein-protein interaction domains such as SH-3 domains, guanylate kinase (GUK) domains, and PDZ domains [118, 119]. The adaptor proteins include members of the membrane-associated guanylate kinase (MAGUK) [120] and membrane-associated guanylate kinase with an inverted orientation of protein-protein interaction domains (MAGI) families [121, 122]), such as ZO-1, -2, -3, MAGI-1, -2, -3, as well as proteins with one or several PDZ domains, such as PAR-3, PAR-6, and MUPP1 [66, 103, 122]. The adaptor proteins serve as scaffolds to organize the close proximity of the second type of plaque proteins, the regulatory and signaling proteins. These include small GTPases, their regulators, and the transcriptional regulator ZO-1-associated nucleic acid binding protein ZONAB (Fig. 4.6;

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for further literature, see [56]). Furthermore, a new protein called JACOP (junction-associated coiled-coil protein) has been discovered. This protein is found in the tight junction complex of epithelial but also endothelial cells and is suggested to anchor especially the junctional complex to the actin-based cytoskeleton [123, 124]. JACOP has considerable sequence similarity to cingulin, another previously detected peripheral protein at tight junctions [125]. In many cases, the role of the regulatory and signaling proteins in tight junction biology is still poorly understood; and it is to be expected that they are involved in completely different aspects of tight junction biology. The proteins of the PAR-3/aPKC/ PAR-6 complex are most likely involved in the regulation of tight junction formation and establishment of cell polarity, since overexpression of dominant-negative mutants of these proteins leads to delayed tight junction formation [126– 128]. Both JAM-A and PAR-3 localize to cell-cell contacts of endothelial cells [129] and, therefore, it is conceivable that the formation of tight junctions is similarly regulated by JAM-A and the PAR-3/aPKC/PAR-6 complex. Information on the maturation state of cell-cell contacts is required for many cellular events which are regulated by cell density (e.g. proliferation) and transcription factors associated with the cytoplasmic plaque at tight junction provide a direct link between tight junctions and the nucleus [67]. Generally, many of these molecules have not been described explicitly in the BBB endothelial cells so far. Therefore, some of them are not included in Fig. 4.6. However, one can not exclude that in the future they have to be incorporated into the scenario of BBB regulation and maintenance. Indeed, the vast majority of experiments addressing the role of tight junctionassociated proteins for tight-junction biology were performed with epithelial cells. The predominant concern of tight junctions is related to the generation of cellular polarity. Regarding polarity, we have also to consider molecules which were originally detected in invertebrate systems but are now known in vertebrates as well and which will have a yet unknown impact in understanding junctions [56, 124, 130–132]. Our knowledge about the role of these tight junction-associated proteins in tight junction formation in endothelial cells, and in particular those in the BBB, is still limited. However, it is to be expected that the principal mechanisms underlying tight junction formation operate in both cellular systems in a similar way.

4.3.2 Caveolae

Barrier permeability is determined by both the tight junction-controlled paracellular and the caveolae-mediated transcellular permeability. In addition, there is receptor-mediated endocytosis, the morphological basis of which is provided by the clathrin-coated pits and vesicles. The literature on these vesicular structures is huge (see, for example, reviews [133–141]). Caveolae have been suggested as sites of endothelial transcytosis, endocytosis, signal transduction, and as docking


Fig. 4.6 Simplified scheme of the molecular composition of endothelial tight junctions. Occludin and the claudins are the most important membranous components, which are proteins with four transmembrane domains and two extracellular loops. The junctional adhesion molecules (JAMs) and the endothelial selective adhesion molecule (ESAM) are members of the immunoglobulin superfamily. Inside the cytoplasm, there are many associated proteins which partly contain PDZ domains binding the C-terminus of the intramembrane proteins (first-order adaptor proteins). Among those are the zonula occludens proteins 1–3 (ZO-1, ZO-2, ZO-3) and the calcium-dependent serin protein kinase (CASK). The three PDZ domains of these proteins are indicated by the pits in the molecular symbols. Among the second-order adaptor molecules, cingulin was described to be expressed in BBB endothelial cells and the junction-associated coiled-coil protein (JACOP) in other endothelial and epithelial cells. Signaling and regulatory proteins described in endothelial cells, but not in any case explicitly in BBB endothelial cells, are the multi-PDZ-protein 1

(MUPP1), the partitioning defective proteins3 and -6 (PAR-3/6), the membrane-associated guanylate kinase with an inverted orientation of protein-protein interaction domains (MAGI), the ZO-1-associated nucleic acid binding protein (ZONAB), afadin/AF6, and the regulator of G-protein signaling, RGS5. All these first- and second-order adaptors and regulatory/signaling proteins control the interaction of the membranous components with the actin/vinculin-based cytoskeleton. Whereas tight and adhesion junctions in epithelial cells are strictly separated from each other, these junctions are intermingled in endothelial cells. The most important molecule of endothelial adhesion junctions is the vascular endothelial cadherin (VE-cadherin). Also, the plateletendothelial cell adhesion molecule (PECAM) mediates homophilic adhesion. Linker molecules between adhesion junctions and the cytoskeleton are primarily the catenins (a-, b-, v-catenin), but also desmoplakin and p120ctn. For further information and for literature, see text.

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sites for glycolipids and glycosylphosphatidylinositol-linked proteins (for literature, see [136, 142]). Among the components of the caveolar membranes are the receptors for LDL (not only present in coated pits), HDL, transferrin, insulin, albumin, ceruloplasmin and advanced glycation end-products (AGE), interleukin-1, vesicle-associated membrane protein-2 (VAMP-2), and caveolin-1/-2. Regarding the blood-brain barrier, caveolin-1 was not only detected in endothelial cells, but also in astrocytes and pericytes [143]. Caveolin-1 binds to cholesterol and fatty acids and forms high molecular weight oligomers. Among the molecules forming signaling complexes at caveolin-1 as a multivalent docking site are heterotrimeric G-proteins, members of the MAP kinase pathway, src tyrosine kinase, protein kinase C, and the endothelial NO synthase. All these molecular complexes are organized in lipid-based microdomains or rafts (as summarized in [140]). The involvement of caveolin-1 at least in NO and calcium signaling processes was impressively documented in the caveolin-1-deficient mouse [144]. The function of caveolae as organelles of endocytosis still remains controversial. Internalization of caveolae has been demonstrated to be regulated by phosphorylation [142]. However, caveolin-1-deficient mice seem to have an unaffected transendothelial transport [144]. Caveolin-1 was proposed not to be required for endocytosis but would stabilize endocytic raft domains decreasing endocytosis [145]. In the endothelium, the relationship of paracellular and transcellular permeability is of crucial importance for the regulation of overall transendothelial permeability (Fig. 4.7). It is well known that the vascular endothelial growth factor (VEGF) is identical to the permeability growth factor (VPF) [146]. VEGF plays a central role in triggering angiogenesis and vascular permeability. It was shown previously that endothelial cells under the influence of VEGF form fenestrations, caveolae, and caveolin-1- and VAMP-positive vesiculo-vacuolar organelles (VVOs) [133, 147, 148]. More recently, the src-suppressed C-kinase substrate (SSeCKS) in astrocytes has been reported to be responsible for the decreased expression of VEGF and increased release of the anti-permeability factor angiopoietin-1 (Ang-1) [149] (see also Chapter 2). In parallel, the authors demonstrated that SSeCKS overexpression increased the expression of tight junction molecules and decreased paracellular permeability in endothelial cells. Indeed, VEGF has been shown to induce the phosphorylation of occludin and ZO-1 which could result in both dissociation of caveolin from the junction [78] and targeting to the luminal membrane. The VEGF receptor 2, also known as Flk-1, is closely associated with caveolin-1, the main molecule of caveolae [150], which has also been shown to co-precipitate with occludin (Fig. 4.7) [151]. In addition, the monocyte chemoattractant protein 1 (MCP-1) was reported to alter the expression of both tight junction-related proteins and caveolin-1 [152]. Thus, tight junctions could play a role as a “sink” for caveolin-1, or, put the other way, occludin-bound caveolin-1 may be a stabilizator of tight junctions. Once dissociated from occludin, caveolin-1 could increasingly bind to the dystrophin-dystroglycan complex, in particular to NO synthase [153], which, at least in muscle cells, is associated with syntrophin [154]. However, any information about a connection between NOS and endothelial dystroglycan is lacking so far. In contrast,


Fig. 4.7 Schematic view of the distribution of some transporters, receptors, and other molecules at the BBB. A = Astrocyte, E = endothelial cell, N = neuron, M = microglial cell, P = pericyte, PC = perivascular cell, AS = A-system of amino acid transport, as present in the abluminal membrane, LS = L-system of the amino acid transport, as present in both the luminal and abluminal endothelial membrane, RMT = receptormediated transport, TFR = transferrin receptor, IR = insulin receptor, Pgp = P-glyco-

protein, DDC = dystrophin- dystroglycan complex, Kir4.1 = inward rectifying potassium channel 4.1, VEGFR = vascular endothelial growth factor receptor, Cav = caveolin, OAP = orthogonal arrays of particles in the glial endfoot membrane, representing the site of the water channel protein aquaporin-4 (AQP4), GJ = gap junctions between astroglial cells, TJ = tight junctions between endothelial cells or cell processes. The gray line around endothelial and perivascular cells represents the basal laminae.

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endothelial NO synthase is present in BBB endothelial cells and its activity is increased in permeable blood vessels [155, 156]. Also, NO donors were shown to disrupt the BBB [157]. Accordingly, the well known cytokine erythropoietin seems to protect the BBB against VEGF-induced permeability by reducing the level of NO synthase and by stabilizing tight junctions [158]. This relationship could suggest that tight junctions and vesicular transcytosis are interconnected inversely. However, as has been demonstrated by means of direct electron microscopical observations, the cleft index, as a morphological indicator of the tight junctional integrity, in capillaries of C6 or RG2 rat glioma was higher than in normal brain tissue. In contrast, the vesicular density was found not to be increased in the tumor. Interestingly, administration of bradykinin or leukotriene C4 was not able to alter the cleft index in both normal brain and brain tumor, but the vesicular density was increased in the tumor only [159], suggesting that vesicular transcytosis and tight junctions can be regulated separately.

4.3.3 Transporters in the Blood-Brain Barrier Endothelium

The tight junction-based paracellular impermeability of the brain capillary endothelial cells implies that the hydrophilic substances essentially needed for the metabolism of the brain require transport through the endothelial wall. Several independent carrier systems for the transport of hexoses (glucose, galactose), neutral, basic and acidic amino acids, monocarboxylic acids (lactate, pyruvate, ketone bodies), purines (adenine, guanine), nucleosides (adenosine, guanosine, uridine), amines (choline), and ions have been described (for recent overviews, see [160, 161]). Among these transporters, the glucose transporter is of special importance due to the fact that glucose is the main energy source of the brain (Fig. 4.7). The 55-kDa form of the glucose transporter isoform Glut1 as one of five members of a supergene family of sodium-independent glucose transporters is highly restricted to the capillary endothelial cells in the brain [160, 162, 163]). This restriction to endothelial cells and the asymmetrical distribution in the endothelial cell membranes (the density of transporter molecules is three to four times higher in the abluminal than in the luminal membrane [164–167]) are believed to reflect the onset of BBB function during development. In functional terms, the lower concentration of transporter molecules in the luminal membrane limits the intensity of glucose flux from the blood to endothelium; and the higher concentration in the abluminal membrane may reduce the endothelial glucose concentration in comparison to that in blood and secure a better efficiency of transport from endothelium to brain parenchyma [166]. In addition to carrier-mediated transport from blood to brain, receptormediated transport has also been investigated intensively. In BBB endothelial cells, there are receptors for the endocytosis of transferrin [168–170], LDL [171], immunoglobulin G [172], insulin, and insulin-like growth factor (Fig. 4.7) [173]. In the case of the insulin receptor and the insulin-like growth factor receptor, it


should be stressed that the BBB of mice (the endothelial cells of which were specifically deficient for both receptors) was not compromised, indicating that neither insulin nor insulin-like growth factor are required for the development and integrity of the BBB [174]. The transfer of dopamine between brain and blood is restricted by the enzymatic conversion of L-DOPA to dopamine by the DOPA-decarboxylase within the endothelial cell (“metabolic BBB”) [175]. Other enzymes such as c-glutamyltranspeptidase and antioxidant enzymes (glutathione peroxidase, glutathione reductase, catalase, superoxide dismutase) are involved in protecting the brain against oxidative stress. Thereby, the reduced glutathione form GSH reacts nonenzymatically, or catalyzed by glutathione peroxidase, with reactive oxygen species and forms the oxidized form glutathione disulfide (GSSG). By the glutathione reductase reaction, GSH can be regenerated from GSSG [176]. It has been shown that oxidative stress induced by cerebral ischemia disrupts the BBB and that antioxidants such as superoxide dismutase and catalase attenuate the BBB disruption [177, 178]. Presumably, the antioxidative capacity of the brain may be promoted by the homeostasis of glutathione metabolism in the brain and between brain and blood. In capillary endothelial cells and in astrocytes, a sodium-dependent GSH transporter has been described which may contribute to the delivery of GSH from blood to brain [179, 180]. There are still other antigens associated with the BBB. For example, in order to restrict free diffusion of lipophilic compounds from blood to brain, a control mechanism is required to export them effectively out of the brain endothelium. In this context, the P-glycoprotein which is a transmembrane protein conferring multidrug resistance is of special importance (see Chapter 19). P-glycoprotein and the multidrug resistance-associated protein Mrp1 [13] have been identified in brain endothelial cell membranes (Fig. 4.7). These proteins importantly are responsible for the active extrusion of nonpolar molecules out of endothelial cells and are therefore in the focus of research on drug delivery in the brain. Interestingly, P-glycoprotein was described to be co-expressed together with caveolin in brain microcapillary endothelial cells [181, 182]. However, only a fraction of P-glycoprotein may be associated with caveolin-1, suggesting that the expression of both proteins may be under the control of defined metabolic requirements [183]. Moreover, another important class of surface molecules mediating the interaction between blood cells and endothelium represents the adhesion molecules. The expression and upregulation of the intercellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1, respectively) have been identified as essential steps for lymphocyte recruitment during inflammation (for a review, see [106]). The transendothelial migration of lymphocytes was shown to be dependent on Rho signaling: when activation of Rho as a consequence of leukocyte binding to the adhesion receptor ICAM-1 was blocked by C3-transferase, leukocyte transmigration was compromised [184]. Whereas the extracellular domain of endothelial ICAM-1 suffices to mediate T cell adhesion, the cytoplasmic domain is required to mediate transmigration of T cells, probably by inducing Rho-signaling within the endothelial cells [185]. The involvement of tight

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junctions during inflammation is a topic which arouses contradictory discussion in the field. It seems widely accepted that inflammatory cells invade the CNS by using the paracellular pathway [102]. However, there is a multitude of morphological evidence that suggests consideration of the transcellular transmigration as well (for reviews, see [56, 105, 106]). This does not rule out that the molecular composition of tight junctions can be altered during inflammation [186, 187].

4.4 Brief Consideration of the Neuroglio-Vascular Complex

The neuroglio-vascular complex is involved in the regulation of blood flow and nutrient supply within the CNS (Fig. 4.7). This regulation includes: (a) control of perfusion parameters differentially realized in specific brain regions according to local requirements, (b) maintenance of energy supply from blood to neuronal and synaptic metabolism via glial cells, and (3) the protection of the nervous parenchyma from alterations in blood composition, particularly from neurotoxic compounds including reactive oxygen species. The network of neurogliovascular interactions (the “neurovascular unit”) is involved in manifold metabolic dependencies between neurons and glial cells on the one hand and between glial cells and vascular cells on the other hand (for recent comprehensive overviews on the neurovascular unit, see [188, 189]). The direct interface between the neuroglial compartment and the vascular compartment is established by the perivascular glial endfeet, forming the glial limiting border. It is generally accepted now that astrocytes play a decisive role in the maintenance of the barrier properties of the brain microcapillary endothelial cells. An interesting correlation exists between astroglial differentiation and BBB maturation. The astroglial differentiation can be morphologically recognized as the polarization of astrocytes, which arises concomitantly with the maturation of the BBB [190, 191] and is not maintained by reactive [192] or cultured astrocytes [193, 194]. Polarization of astrocytes means getting a molecular and structural heterogeneity of specific membrane domains of the astroglial surface. Where the processes of glial cells (so-called endfeet) contact the superficial or perivascular basal lamina (glia limitans superficialis et perivascularis), the glial membrane is studded with numerous square arrays or orthogonal arrays of intramembranous particles (OAPs) which can only be visualized by means of the freeze-fracture technique (for an overview of the literature, see [193]; Figs. 4.7 and 4.8). Where the contact of the glial cell membrane with the basal lamina is lost by bending away into deeper parenchymal regions of the neuropil, the density of OAPs is dramatically reduced. In highly polarized epithelial cells, the apical and basolateral membrane domains are separated by tight junctions; but this is not the case in highly polarized astrocytes. The establishment and maintenance of the glial polarity is suggested to be evolved by the extracellular matrix of the basal lamina (Fig. 4.8). Regarding the OAPs, it is well known now that they contain at least the water channel protein aquaporin-4 (AQP4) (Fig. 4.7). Aquaporins mediate water move-


Fig. 4.8 Working hypothesis on glial involvement in the formation and maintenance of the BBB. When the BBB is fully established, the associated astroglial cells are highly polarized in terms of orthogonal arrays of particles (OAPs, symbolized as black rectangles; picture in the middle of the scheme is a freeze-fracture replica) and the water channel protein aquaporin-4 (AQP4). The development of astroglial polarity goes along with an increasing expression rate of the heparansulfate proteoglycan agrin within the perivascular basal lamina. In culture and under pathologic conditions such as brain

tumor, agrin is down-regulated and the OAPs are redistributed (black rectangles), or lost, or AQP4 is no more recognizable in freeze-fracture replicas (white rectangles). This down-regulation may be causative for the redistribution of AQP4 across the whole surface of the glial cell, which can be described as a loss of glial polarity. This loss can constitutively be observed when the BBB is disturbed. Therefore, agrin as a molecule significantly involved in the clustering of the dystrophin/dystroglycan/AQP4 complex seems to play a pivotal role in the management of the endothelial BBB (see text).

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ments between the intracellular, interstitial, vascular, and ventricular compartments which are under the strict control of osmotic and hydrostatic pressure gradients (see Chapter 9) [195–198]. The involvement of AQP4 in OAP formation was demonstrated by the absence of OAPs in the astrocytes of an AQP4-deficient mouse [199], by the formation of OAPs in chinese hamster ovary cells stably transfected with AQP4 cDNA [200], and by the immunogold fracture-labeling technique showing that AQP4 is a component of the arrays [201, 202]. Moreover, Nielsen et al. [203] were able to demonstrate by immunogold immunocytochemistry that the distribution of the AQP4-related immunoreactivity was identical to that of the OAPs. Interestingly, the OAP-related polarity of astrocytes seems to correlate with the quality of the BBB (Fig. 4.8) [190, 193]. An indirect proof of this relationship is the observation that, under brain tumor conditions when the BBB is known to be leaky, the OAP-related polarity of glial cells was decreased (Fig. 4.8) [204]. In addition, the AQP4 content as detected by immunocytochemistry was increased [205, 206]. The apparent contradiction of upregulation of AQP4 and downregulation of AQP4-positive OAPs in glioma cells can be resolved by the assumption that, under glioma conditions, AQP4 exists separately from OAPs in the glial membrane and is no longer restricted to the glial endfeet membranes. Indeed, AQP4 exists as two isoforms (M1, with 323 amino acids, and M23, with 301 amino acids [207]). Only isoform M23 seems to assemble in OAPs, whereas isoform M1 does not [208]. This could mean that, under pathological conditions, the ratio between the M1 and M23 isoforms of AQP4 changes heavily in favor of M1. However, this seems not to be the case, again supporting the view that M23 would become unrecognizable in freeze-fracture replicas by dissociating from AQP4-based arrays (Warth and Wolburg, unpublished observation). What this would mean in functional terms is unknown so far. In any case, the restriction of AQP4 immunoreactivity at the endfoot membrane was maintained only where agrin was present in the perivascular basal lamina [206]. Agrin is an extracellular heparan sulfate proteoglycan which was originally characterized as the essential molecule for clustering acetylcholine receptors at the motor endplate [209, 210] but has also been described as being important within the CNS, particularly for the integrity of the BBB [211–213]. The agrin splicing variant Y0Z0 was reported to be specifically present in the endothelial cell basal lamina of CNS capillaries [214]. If agrin was absent from the basal lamina, AQP4 immunoreactivity was randomly found across the whole surface of the cell [215]. This would suggest that agrin is responsible for the restriction of AQP4 molecules at the glial endfoot membrane (Fig. 4.8). However, agrin has no binding site to AQP4, but to a-dystroglycan [216]. aDystroglycan is a member of the dystrophin-dystroglycan complex (DDC) which is best investigated in the muscular system, but is also present in the CNS (Fig. 4.7) [154, 217]. Thus, a-dystroglycan has to be connected to AQP4. Indeed, they are found to be coexpressed in glial cells. Another member of the DDC is a1-syntrophin, which has a binding site to dystrophin and a PDZ domain binding to AQP4 [218]. In glioblastoma, the binding strength between a1-syntrophin and AQP4 seems to be strong enough to bind AQP4 and a1-syntrophin together


during their redistribution across the surface of the glioma cell [206]. In contrast, dystrophin remains restricted at the endfoot membrane suggesting a cleavage of the dystrophin/a1-syntrophin/AQP4 complex. Concomitant with these observations, we found a loss of a-dystroglycan in the perivascular domain of glioma cells [206]. Thus, loss of agrin went along with loss of a-dystroglycan, redistribution of AQP4/a-syntrophin and loss of OAPs, including a severe reduction in the OAP-related polarity of glioma cells. All these observations together speak in favor of the suggestion that the OAPs consist of more than AQP4, but contain components of the DDC as well. It has been shown that the truncated dystrophin isoform Dp71 is essential for the clustered localization of the weakly rectifying potassium channel Kir4.1 in retinal Müller (glial) cells [219]. In addition, the PDZ domain of a-syntrophin can also bind to Kir4.1 [220], which is normally restricted to the endfoot membrane in astrocytes and retinal Müller glial cells (Fig. 4.7) [221–223]. On the basis of colocalization of AQP4 and Kir4.1 in retinal Müller cells and due to the well known fact that water fluxes are driven by ion fluxes, it was hypothesized that K+ clearance is coupled to water flux [189, 203, 224–227]. Accordingly in the a-syntrophin-deficient mouse, in which AQP4 is dislocalized across the glial surface, the K+ clearance was delayed [228]. The authors argue that K+ uptake would be facilitated if accompanied by water flux. In the hypoxic retina, Kir4.1 is downregulated in retinal Müller cells compromising the spatial buffering capacity; and as a consequence, intracellular K+ concentration increases and consecutive water flux is causative for the observed cell swelling [226, 227]. Alternatively, the cell swelling can also be due to a failure to release water at the endfoot membrane, because the molecular complex consisting of Kir4.1 and AQP4 may dissociate. In human glioblastoma tissue, we were able to observe a redistribution of both anti-AQP4- and anti-Kir4.1-immunoreactivities across the surface of the glioma cell [229]. The observation that Kir4.1, but not AQP4, was already redistributed in low-grade astrocytomas suggested different targeting or cluster mechanisms for Kir4.1 and AQP4. An uncoupling of water transport from K+ siphoning under different pathological conditions, such as brain contusion, bacterial meningitis, and brain tumors, has recently been postulated [230] and may be one reason for cell swelling. Interestingly, before the detection of aquaporins, OAPs were frequently discussed as morphologically correlating to potassium channels [193, 231–233]. However, we must not forget that this kind of molecular aggregation of different channels in one morphological structure is also established at the subpial endfoot membrane and not exclusively at the perivascular endfoot membrane, suggesting that the OAP-related molecular arrays have no exclusive significance for the BBB. In the a-syntrophin-deficient mouse, Amiry-Moghaddam et al. [234] described an elongation of the survival time of animals under hyponatremic conditions in comparison to wild-type mice. The reason for the longer survival time might be the reduced formation of brain edema, which was interpreted as a consequence of the redistribution of AQP4 after its cleavage from a-syntrophin. However, as we were able to show, the cleavage of a-syntrophin from AQP4 seems not to be

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a prerequisite for the redistribution of AQP4 [206]. Rather, we believe that loss of agrin reduces the OAP/AQP4-related polarity of astrocytes. The loss of agrin might be caused by an activation of metalloproteinase-3, which has recently been reported to occur under conditions of cerebral ischemia [235]. Furthermore, this could lead to a redistribution of “free” AQP4 molecules outside the OAP structure, or an upregulation of the M1 isoform of AQP4 (see above), a downregulation of Kir4.1, and a consecutive failure of spatial buffering of K+, followed by osmotic water influx and swelling. All these processes together might represent key events for both the development of an edema and the loss of the glial cell ability to maintain BBB properties within the endothelial cell. An unpolarized astrocyte has already been suggested to be unable to induce or maintain the complete set of BBB properties in endothelial cells (Fig. 4.8) [236].

4.5 Conclusions

It has been 120 years since the discovery of the BBB by Paul Ehrlich. In the following period, BBB research has focused on the morphological description of the barrier, using mainly conventional histological and electron microscopical methods as well as methods to demonstrate the tightness of the barrier against a variety of low and high molecular weight substances (for a review, see [237]). Tight junctions have been described as a network of protein particles, using freeze-fracture electron microscopy. But, it required almost a further 30 years and a great methodological advance in molecular biology to discover the protein components of the tight junctional complex and to identify the protein particles within the freeze-fracture replicas. Then, it took further time to recognize these tight junctional complexes as very dynamic structures. Now, it might be possible to go into more detail, to characterize the interplay of these molecules and their regulation by and association with cellular signaling cascades and cytoskeletal components. It has been shown that the endothelial barrier in the brain differs from epithelial barriers. The simple observation that epithelial, but not endothelial cells are able to form a high-resistance and low-permeability barrier in vitro sheds light on the importance of the brain microenvironment in the formation and maintenance of the barrier in vivo. This microenvironment consists of endothelial cells, pericytes, microglial cells, astrocytes, neurons, and the extracellular matrix in between, which itself forms a microcosmos of its own. All components may or may not operate together or at certain periods during development or during pathological derangements and in different combinations which are not recognizable so far. The greatest future challenge will be characterizing the mechanisms involved in BBB differentiation and pathology in order to understand the key principles of barrier formation in the CNS.

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Acknowledgments

Personal work cited in this chapter was supported by grants from the Fortüne program of the University of Tübingen, the IZKF Tübingen, the Deutsche Krebshilfe, and the Deutsche Forschungsgemeinschaft. Drs. A. Lippoldt and K. Ebnet contributed to this work.

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5 Pericytes and Their Contribution to the Blood-Brain Barrier Markus Ramsauer

5.1 Introduction

The simplistic view of the blood-brain barrier (BBB), which defines this barrier as an exclusively endothelial entity, must be reconsidered. In particular, cerebral pericytes constitute a neglected component of the metabolic BBB complex. These cells are ideally positioned to create an interface between the circulatory system and the brain parenchyma. Pericytes, originally described in 1870 by Rouget, are perivascular cells that are found adjacent to capillaries, share a common basement membrane with the cerebral endothelium, and are distinctively shaped with many cytoplasmic processes that encircle capillaries. These cells are derived developmentally from the mesoderm and are indicated to possess morphological and functional differences within the same tissue or even within a single capillary bed. Pericytes are difficult to define, plastic, and have the capacity to differentiate into other mesenchymal cell types. Thus, these cells in the brain may serve multiple functional roles and among these are macrophage activity, modulation of blood flow and vascular permeability, and regulation of vessel growth and stability. Contraction (and reciprocal relaxation) appears to be the way that pericytes influence microvascular blood flow. Furthermore, pericytes in the central nervous system (CNS) express macrophage functions and are actively involved in immune responses operating as a “second line of defense” at the BBB. And finally, they play a regulatory role in BBB differentiation, endothelial cell tight junction formation, and brain angiogenesis. The regulation of endothelial cells has been suggested to control vessel growth and contribute to vascular stability. Pericytes have been reported to provide: (a) BBB-specific enzymes, (b) potential modulators of endothelial permeability, (c) stabilizing effects on microvessel walls, and (d) a promoting activity on angiogenic processes and capillary sprouting. A host of different cell factors and signaling agents appear to be involved with these cellular functions, some effecting the pericyte and others produced by this cell. What is emerging is a realization of the reciprocal dependence of the periBlood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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cyte and its nextdoor neighbor, the endothelial cell, and the resulting complex biological feedback mechanisms.

5.2 Pericyte Structure and Positioning

The BBB regulates the exchange of blood solutes with the interstitial cerebral fluid and consists of a physical and metabolic barrier. The BBB is composed of the microvascular endothelium lining blood vessels surrounded by pericytes and astrocytes, which tightly clasp cerebral microvessels by their endfeet (Fig. 5.1 a). Due to their strategic position, pericytes create an interface between the circulatory system and the surrounding tissue (CNS; [1]). To date, pericytes have been defined primarily by their periendothelial cell location in the microvasculature. A variety of terms are used as synonyms for pericytes: microvascular smooth muscle cells, perivascular macrophages, (intra)mural cells, myofibroblasts, and perivascular cells. However the term pericytes best describes their anatomic relevance (peri: around, cyto: cell). Pericytes are intimately associated with endothelial cells. An intercellular distance of less than 20 nm is often noted between them [2]. Pericytes are enveloped in a basement membrane, which is continuous with the basal lamina of the capillary tubes. The thin basement membrane provides mechanical support and a barrier function. There is in vitro evidence that both pericytes and endothelial cells contribute to the production of this basement membrane [3, 4]. Being contained within the basement membrane distinguishes them from smooth muscle cells, which are separated by the basement membrane from endothelial cells and do not establish direct contact. In mature vessels, the most reliable identification is still electron microscopy, revealing the complete surrounding of the pericytes by the endothelial basement membrane. However, in embryogenesis and during angiogenic sprouting, this method of identification cannot be applied, since the basement membrane is not fully developed [5]. Each pericyte possesses a large cell body with a prominent nucleus and a small amount of surrounding cytoplasm [2]. In the CNS, pericytes have an oval to elongated cell body arranged parallel to the vessel long axis. The shape and contour of the cell body is determined by the vessel outline and the overlying basement membrane. Protruding from the soma are several long primary processes, which also run parallel to the long axis of the blood vessel; and orthogonally oriented smaller secondary processes radiate along the length of the primary process and encircle the capillary wall. This provides the appearance that pericytes cradle or girdle the blood vessel [6]. Early studies by Zimmermann [7] revealed the presence of up to 90 processes with a width of 0.3–0.8 lm per 100 lm of capillary length. Interestingly, the distance between cell bodies is up to 50 lm in the brain, suggesting an interdigitation of processes among neighboring pericytes. Processes from multiple pericytes can cover a single endothelial cell and a single pericyte can extend processes to more than one capillary

5.2 Pericyte Structure and Positioning

Fig. 5.1 (a) This schematic view recapitulates the in vivo situation of the BBB (EC = endothelial cells, PC = pericytes, AC = astrocytes, BM = basement membrane). (b) Immunogold transmission electron micrograph from a cross-section of an isolated cerebral microvessel, utilizing pericytic aminopeptidase N. The pericyte (PC), identified by the ensheathing basement membrane, shows immunogold beads on its abluminal plasma membrane, whereas the endothelial cell (EC) is devoid of immuno-

reactivity. (c, d) Pericytes in vitro grow not contact-inhibited with overlapping processes (c) and exhibit noticeable retraction with formation of numerous multicellular nodules (d). (e, f) Double-immunofluorescence of mixed endothelial-pericyte cultures in triculture with primary astrocytes. Endothelial cells are visualized by immunostaining for vW F VIII-ra (red). Capillary-like structures closely associated with pAPN-positive pericytes (green, e) are surrounded by GFAP-expressing astrocytes (green, f).

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within the microcirculation [1]. Their processes penetrate the basement membrane to directly contact the underlying endothelium and, in a reciprocal manner, endothelial processes penetrate into the pericytes [8]. Also, reciprocal invaginations may occur in endothelial cells or pericytes, filled by processes of the other cell. These have been described as heterologous “peg and socket” contacts [9]. Next to an array of adhesion plaques that exhibit membrane specializations between pericytes and endothelial cells [2], the two also form gap junctions [10, 11]. Gap junctions provide direct connections between the cytoplasm of the two cells and allow the passage of ionic currents and small molecules [12]. These contact sites are thought to provide anchorage and to support transmission of a mechanical contractile force from the pericytes to the endothelium [2, 13]. The pericyte coverage of the abluminal endothelial surface is only partial, varies extensively between the capillary beds of different tissues, and may exhibit vessel variation [14]. The highest pericyte density has been described for neural tissues and is stated to be almost in parity in brain and retina. Pericyte to endothelial ratios are 1 : 3 in the brain and only outnumbered in the retina (1 : 5). For comparison, pericyte to endothelial ratio is stated to be 1 : 100 in striated muscle [15]. A comparison of pericyte coverage (pericyte plasma membrane length in contact with the vascular circumference, versus the outer circumference of the endothelial cell tube) in the rat’s retina and brain revealed a ratio of 0.41 for the retinal microvessels and ranged from 0.22 to 0.30 for five regions of the cerebral cortex [16]. The high number of pericytes may reflect their contribution to the blood-retina and blood-brain barrier. The number of pericytes, however, varies significantly among different-sized vessels, being found on arterioles, venules, and particularly capillaries [1]. On either side of a capillary bed, there is a continuum of cells, from “true” pericytes surrounding capillaries, via “intermediate” cells resembling both smooth muscle cells and pericytes at the interface between arteriolar or venular capillaries and arterioles or venules, to “true” smooth muscle cells surrounding terminal arterioles, venules, and larger vessels. The smooth muscle cells provide structural support to the large vessels and are, especially in the arterioles, important regulators of blood flow due to their contractile behavior. Pericytes are more abundant on post-capillary venules and relatively sparse on capillaries. The distribution of pericytes could reflect postarteriolar hydrostatic pressures [17]. In addition to this selective positioning in the vessel wall, there are structural differences among pericytes on the arterial and venous sides of capillary beds. Pericytes in “true” capillaries are often highly elongated with extensive slender processes that wrap many times around the endothelial tube, whereas in the post-capillary venules the pericytes are stubby with thick and short radial processes [15].

5.4 Pericytes in Culture

5.3 Pericyte Markers

So far no pericyte-specific marker has been identified, such as the von Willebrand factor VIII-related antigen (vWFVIII-ra) for endothelial cells, which makes pericytes positive identification often difficult. However, a number of pericyte markers have been described which can be used to differentiate them from other cell types. Cerebral pericytes express surface markers: high molecular weight melanomaassociated antigen (HMW-MAA; called nerve/glial antigen 2, NG2, in the mouse; [18, 19]) and differentiation antigen Thy 1.1, found on thymus-derived lymphocytes, is also expressed by brain pericytes and astrocytes [20]. A monoclonal antibody (3G5) directed against a surface glycolipid has been shown to be a useful marker for pericytes [21]. Rat pericytes also express the integrin-subunit CD11b (aM) and low levels of the marker ED2 [22, 23]. Although they share markers with cells of macrophage lineage, they do not express CD45 and isolectin IB4 (GSA; [23]). Krause et al. [24] reported the characterization of a monoclonal antibody that recognizes a 140-kDa protein specific for pericytes of the rat. Later, the protein was identified as a specific isoform of aminopeptidase N (CD 13) in cerebral pericytes (pAPN; [25]). The lack of pAPN expression in brain regions devoid of a tight endothelium, like the circumventricular organs, suggested that this ectopeptidase is a member of the metabolic complement of the BBB which is involved in neuropeptide degradation [26]. pAPN was recently revealed to be a late BBB marker in rodent brain development, which occurs around day E18 of brain angiogenesis [27] and is expressed at the extracytoplasmic side of the pericyte plasma membrane (Fig. 5.1 b). Cerebral pericytes express vimentin as an intermediate filament but not desmin, which is found in peripheral pericytes and smooth muscle cells [28]. Pericytes contain both smooth muscle and nonsmooth muscle isoforms of actin and myosin [29]. In situ, pericytes from “true” capillaries (mid-capillaries) are reported to be virtually devoid of smooth muscle isoforms, whereas pericytes from pre-capillary arterioles and post-capillary venules contain mainly smooth muscle isoforms [30]. The expression of these markers varies, depending on the species-, tissue- or development-related contexts. For example, in chicken embryos, pericytes surrounding the angiogenic vasculature of the brain can easily be visualized by their expression of smooth muscle actin (SMA) [31]; but this is not the case in mice or rats [32].

5.4 Pericytes in Culture

Most of the data about pericytes is generated from in vitro experiments. Pericyte isolation and culture techniques are usually modifications of a protocol worked out by Gitlin and D’Amore [33]. This involves mechanical disruption of tissue, enzymatic digestion, and collection of capillary fragments that contain both en-

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dothelial cells and pericytes. The vascular cells can be differentiated by their preferential growth on different culture substrata. Endothelial cells have been noted to prefer coatings with some matrix component [34]. When grown under supplemented culture conditions (10% fetal bovine serum) on uncoated plastic, the proliferation of pericytes is favored over the other cell species. Pericytes, unlike endothelial cells, generally grow at similar rates regardless of the substrates [15]. Fetal bovine serum contains sufficient growth factors, such as platelet-derived growth factor (PDGF), to stimulate pericyte proliferation and to yield almost pure cultures of pericytes. Since endothelial cells are inhibited in growth by proliferating pericytes [35, 36], the growth of endothelial cells is rare and transient in primary cultures. Proliferation of pericytes is favored and they eventually become the dominant cell type. Cultured pericytes appear as large, spreading, stellate cells with highly irregular edges (Fig. 5.1 c). Pericytes in culture display numerous long filamentous processes and are characterized by prominent intracellular fibers of actin (stress fibers). They grow in small islets, proliferate slowly with a prolonged lag phase, and do not establish true monolayers. Once in the log phase of growth, the doubling time is in the order of 3–4 days. Their morphology differentiates them from endothelial cells (“cobblestone-like” morphology), fibroblasts (long, spindle-shaped, with extended filopodia), and smooth muscle cells (“hill and valley” growth). Their growth is not contact-inhibited and, after reaching confluence, pericytes form multilayers and then nodules by a process involving the sudden retraction of a multilayer area into a small nodule or cell aggregate, leaving empty patches in the substratum, re-attachment of the nodule on the substratum, followed by cell migration out of the nodule and cell proliferation (Fig. 5.1 d) [37]. Nodule formation is associated with changes in the synthesis and distribution of matrix molecules and extracellular calcification occurs in the nodules [38]. Thus, pericytes in vitro can differentiate along the osteogenic pathway. Culture conditions may influence pericyte phenotype and marker expression. Alpha-smooth muscle actin (a-SMA) protein is expressed in pericytes in vitro but not in brain microvascular pericytes in vivo [39]. The expression and organization of a-SMA can be modulated by the presence of transforming growth factor-beta 1 (TGF-b1) [40], endothelin-1 (ET1) [41], and extracellular matrix molecules [42]. Such factor-dependent phenotypic changes may be the basis for pericyte functional heterogeneity [28]. In order to gain more information on the inductive mechanisms involved in the expression of pAPN, a prominent pericyte-specific BBB enzyme, and to obtain purified cultures of cerebral pericytes, we designed a solid-phase isolation technique, which makes use of the monoclonal antibody directed against the pAPN [43]. pAPN has been detected to constitute a major component of the pericytic protease complement and can be regarded as a specific marker for the pericytic phenotype in brain. The isolation protocol consisted of an immunoabsorption of antibody-labeled pericytes to immunomagnetic beads. Expression of pAPN was high in acute isolated pericytes, as revealed by immunocytochemis-

5.5 Contractility and Regulation of Blood Flow

try. In this study, we showed that the pAPN associated with the BBB is downregulated in pericytic cultures. This observation is in accordance with previous data describing comparable in vitro effects for BBB-specific enzymes of endothelial or pericytic origin, such as c-glutamyl transpeptidase (GGT) [44]. Although purified pericytes deprived of endothelial cells did not reveal a re-expression effect, pericytes that were kept in contact with endothelial cells were able to acquire a pAPN-positive phenotype, indicating that endothelial cells constitute an essential requirement for the in vitro re-expression of pAPN. The in vitro history of the pericytes is essential for the re-inducibility of pAPN. Thus, our experimental design provides stringent in vitro evidence that BBB differentiation requires the cooperation of different vascular cellular components and that the achievement of functionally differentiated cerebral pericytes is closely related to endothelial cells. Because of their close association with endothelial cells in vivo, the physiological functions of pericytes are best studied in the context of their interactions with endothelial cells.

5.5 Contractility and Regulation of Blood Flow

It is usually stated that pericytes have a contractile function, although this has been extensively scrutinized and debated for many years. In fact, originally, Rouget [45] regarded pericytes as a variety of smooth muscle cells and this view was adapted later by Zimmermann [7]. Many researchers today presume that the pericytes are contractile elements related to smooth muscle cells and involved in the regulation of blood flow through the microvasculature, although definitive evidence of their contractility remains elusive. The first line of such evidence came from the indication of contractile proteins in the pericytic cells. Pericytes exhibit a cytoskeleton similar to vascular smooth muscle cells. Early ultrastructural studies reported the presence of actinand myosin-like filaments in rat brain pericytes [46]. More recent publications describe the similarities between pericyte and smooth muscle cell actins [29] and (tropo-)myosins [47, 48]. In the pericytes of small capillaries, nonmuscle isomyosin is the predominant form, whereas the smooth muscle isomyosin is present in a very low concentration. The reverse relationship is found in pericytes associated with larger capillaries and post-capillary venules. At the subcellular level, actin is localized in stress fibers and microfilament bundles [49, 50]. Nehls and colleagues [28] reported that muscle-specific actin appeared to be absent or scarce, even in the pericytes of pre- and post-capillary segments. A systematic study has shown that the alpha-actin mRNA is expressed in brain pericytes in tissue culture, but the immunoreactive alpha-actin protein is not expressed in brain microvascular pericytes in vivo [39]. The nonphysiological conditions of tissue culture may cause dedifferentiation of pericytes towards a primitive, smooth muscle-like phenotype. These authors hypothesize that only

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the SMA-expressing capillaries participate in blood flow control and the midcapillaries serve a different function. Functional evidence for the contractile ability of the pericytes comes from studies utilizing in vitro techniques, where their contraction of collagen or silicone substrata has been directly observed [51–53]. However, contraction of collagen and silicone rubber in vitro does not necessarily equate to contraction in vivo and the latter is much more difficult to prove morphometrically. But especially the assessment of cell contraction in vitro allowed the investigation of vasoactive compounds which alter or regulate pericyte contractile tone. Pericytes have been indicated to possess both cholinergic and adrenergic receptors and their response leads to relaxation or produces contraction [6]. It has been demonstrated that the contractile response of pericytes could be altered by certain vasoactive agents; for example histamine and serotonin contracted the pericytes [54]. The most attention received concerns the endothelium-derived vasorelaxants nitric oxide and prostacyclin, the vasoconstrictors angiotensin II and endothelin-1, and their corresponding receptors expressed on pericytes [41, 55, 56]. Through a paracrine mechanism, endothelial cells could control contraction in pericytes, which suggests the involvement of cerebrovascular auto-regulation. Given their intimate association with vessels, their shape, distribution, and morphology, it is compelling to seriously consider possible functions that would involve contractility analogous to the smooth muscle cells of larger vessels. At the level of the capillary, extremely subtle changes in the internal diameter could be functionally very effective at altering capillary blood flow. In the mature vascular system, the endothelium is supported by mural cells, which express characteristics specific to their localization, for example pericytes of the pre- and post-capillary segments display gradual transitions to smooth muscle. Therefore, at least two pericyte populations appear to exist: smooth muscle-like transitional pericytes and nonmuscle-like mid-capillary pericytes [30, 39, 40]. The marked similarity to vascular smooth muscle cells has led to the theory that pericytes and vascular smooth muscle cells represent phenotypic variants of a continuous population of mural cells. Depending on external stimuli, pericytes have been suggested to give rise to smooth muscle cells and vice versa [28]. Thus, pericytes may function as the progenitors for smooth muscle cells in cases of vessel enlargement or remodeling.

5.6 Macrophage Function

Pericytes in different tissues have been suggested to serve different functional roles; and diversity or heterogeneity within the pericytes of an individual tissue has often been noted. The potential phagocytotic role of pericytes has only been well investigated in the CNS, leading to the proposal that pericytes are to be considered part of the BBB [57]. A structural feature often used in the assessment of macrophages is the presence of lysosomes. Pericytes have been shown

5.6 Macrophage Function

to contain notable lysosomes and inclusion bodies, which are strongly reactive for acid phosphatase [58]. Lysosomal inclusions are common in pericytes of the CNS, but are only occasionally observed in other tissues [2]. In further support of the theory that pericytes act as phagocytic cells, the cells ingest substances after systemic injection from the circulation [59], after intraventricular administration [60], and after direct introduction into the extracellular fluid [61]. Based on this activity, it has been suggested that pericytes serve to clean the extracellular fluid. The phagocytic capacity of CNS pericytes has been well documented in numerous conditions of trauma [62–65]. In addition, in vitro ingestion of various materials by the cells, including polystyrene beads, can be directly observed [23]. It appears, however, that pericytes may be limited to the pinocytosis of proteins such as horseradish peroxidase or trypan blue, as no phagocytosis of carbon particles by pericytes (but perivascular cells not completely surrounded by basement membrane) was seen in a recent critical study [66]. With respect to macrophage function, pericytes have been reported to possess numerous marker components of macrophages under some conditions: these include CR3 complement receptor, leukocyte-common antigen CD4, Fc receptor, and classes I and II major histocompatibility complex molecules (MHC class I and II) [67]. Therefore, CNS pericytes may be actively involved in the regulation of leukocyte transmigration, antigen presentation, and T-cell activation, critical aspects of macrophage function [5]. They constitutively express low levels of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) [68], which have potential costimulatory activity in MHC class II-dependent antigen presentation. Like the adhesion molecules, T-cell adherence is increased by TNF-a treatment. Another component which has been demonstrated to up-regulate pericytes is interferon-c [69]. While the pericytes express some macrophage properties, the cells are also capable of up-regulating this activity, like other tissue macrophages. Pericytes do not display full activity in the normal CNS tissue and have been viewed as a “second line of defense” at the boundary between blood and brain [26]. Maxwell and Kruger [62] reported that, following irradiation of the brain, pericytes undergo mitosis and penetrate the brain parenchyma. Similar anatomical investigations led some authors to speculate that pericytes transform into microglial cells [64, 70]. The origin of microglia from elements detached from the walls of the vessels has been sustained throughout the decades, but it has been denied as well. They are distinguished from pericytes by morphology, location, and functional properties. However, while clearly being different cell types, microglia and pericytes do appear to share the same origin and overall functional role. A recent study has shown a strong correlation between the numbers of microglia and pericytes in different tissue regions, suggesting a transformational relationship and, on the basis of electron microscopic images, proposed that pericytes break out of the basal lamina, which involves astrocytes, and become perivascular microglia [71]. At this point, the exact relationship between pericytes and microglial cells still remains incompletely resolved. Furthermore, a subgroup of pericyte-like cells, based upon their not being completely enclosed

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within the vascular basal lamina, has been distinguished from pericytes. These cells reside on the surface of the basal lamina, between it and the glia limitans [66]. Also, the relationship between pericytes and the pericyte-like perivascular cells is unknown. This makes investigation of a release or liberation of pericytes from the basement membrane even harder, as the classic definition of a pericyte as a cell embedded in the capillary basement membrane cannot be applied in these situations. Overall, in vivo and in vitro, many structural and functional properties of macrophages are exhibited by pericytes and they appear to be intermediate in terms of their macrophage potential.

5.7 Regulation of Homeostasis and Integrity

Important aspects of vascular homeostasis are related to the control of vascular tone and perfusion by way of endothelial cell-secreted contracting or relaxing factors and control of the leukocyte traffic from the blood compartment to the interstitium [15]. In addition, an active role of pericytes in the regulation of the BBB was highlighted by their expression of degrading enzymes such as butyrylcholinesterase (BChE) [72], c-glutamyltranspeptidase (GGT) [44, 73], glutamyl aminopeptidase (EAP) [56], and aminopeptidase N [25]. Pericytes thus have been regarded as a “second line of defense”, which helps to maintain homeostasis of the CNS, particularly during breakdown of the endothelial BBB. With respect to homeostasis, to maintain a selective permeability barrier for plasma constituents and to regulate their passage from the intravascular compartment to the interstitium is very important. In this context, the role of pericytes in BBB establishment should be reconsidered. Recent in vitro studies have shown that pericytes and endothelial cells of CNS origin have mutual influence on each other’s biosynthetic and replicative behavior. Pericytes also influence the BBB at the level of the endothelial tight junction and transendothelial vesiculation. BBB differentiation was originally thought to be a process induced and directed by astrocytes [74, 75]. However, astrocytes may not be the only cells to contribute regulating factors, since the development of endothelial cell tight junctions is not altered in conditions associated with astrocyte-endothelial cell decoupling [76] or in areas lacking astrocytes [77]. The group of Dore-Duffy found increased pericyte coverage in glial fibrillary acidic protein (GFAP)-deficient mice characterized by reduced cytoarchitectural integrity of the astroglial layer surrounding the microvasculature [5]. Early in the embryonic CNS development of the mouse, invasion of the intraneural section by the perineural mesenchymal endothelial cells is accompanied by pericytes [78]. The subsequent appearance of tight junctions and the decrease in pinocytotic vesicles are also associated with pericyte coverage. Others have shown that growth and maturation of the microvasculature is dependent on the

5.7 Regulation of Homeostasis and Integrity

establishment of endothelial cell-pericyte gap junctions [11]. Furthermore, there is also recent evidence indicating that pericytes are able to mimic the astrocytic inductive influence. The absence of pericytes in platelet-derived growth factor-B (PDGF-B)- and PDGF receptor-beta (PDGFR-b)-deficient mouse embryos leads to defects in endothelial junction formation and to the formation of numerous cytoplasmic folds at the luminal surface of the endothelium [79]. This suggests that pericytes control endothelial differentiation in vivo. Most in vitro studies of capillary permeability have focused on endothelial cell monolayers and ignored the second cell that forms the capillary wall: the microvascular pericyte. Later, a model to study the permeability characteristics of endothelial cells, pericytes, and co-cultures of both cell types using semipermeable culture inserts was described [80]. Indeed, the addition of pericytes to endothelial monolayers increased their barrier effect to macromolecules and electrical resistance. Other transfilter co-culture experiments using conditionally immortalized adult rat brain pericytes revealed that the pericyte-derived angiopoietin-1 (Ang-1) induces occludin expression, an integral tight junction protein [81, 82]. The results of Sundberg et al. [83] provide strong evidence that Ang-1 is expressed by pericytes in vitro and in vivo and that the role proposed for Ang-1 in vessel maturation in development can be extended to vessel maturation after angiogenesis in adult tissues. Only recently it was shown in another co-culture model that brain pericytes induce and enhance the barrier function and P-glycoprotein (P-gp) functional activity of brain endothelial cells [84]. This pericyte-induced up-regulation of BBB properties was mediated, at least in part, through continuous production of transforming growth factor-beta 1 (TGF-b1). Another, and more speculative, possible function of pericytes is that they may be modulators of leakage from microvessels [2]. Pericytes are most numerous on venules, the most leaky microvessels. Pericytes and their basal lamina appear to block the extravasation of macromolecules and blood-borne elements and provide mechanical support. Pericytes synthesize and deposit various extracellular matrix proteins, such as laminin, fibronectin, collagen (types I, III, IV, X), tenascin, and thrombospondin [37]. Behaving as a mechanical part of the vessel wall was one of the originally stated functions of pericytes. Maintenance and structural rigidity of the microvessel wall could also be inferred by the presence of stress fibers within pericytes, the enveloping design of pericytes, and pericytic contacts with endothelial cells that provide anchorage and are also thought to support transmission of the mechanical contractile force from the pericytes to the endothelium. Additionally, pericyte loss observed in PDGF-deficient mice results in compromised vascular stability; and these mice also develop capillary microaneurysms [85]. All in all, the pericyte is in a prime location to be involved with changes in microvascular permeability and to contribute to stable and mature capillaries.

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5.8 Angiogenesis and Stability

Development of the vascular system is a complex process, governed by vascular endothelial growth factor (VEGF), that begins with the clustering of endothelial precursors, also known as angioblasts, into tube-like endothelial structures (vasculogenesis) [86]. Vascularization of neural tissues, like the brain, occurs by angiogenesis [74]. The preexistent vessel tubes hereby remodel and form the more complex architecture of the vascular system. Vessel assembly, patterning, and maintenance are complex and highly regulated processes. Developmental studies suggest that the endothelial cells in the nascent tubes may govern the acquisition of additional vessel layers [87]. As the next few paragraphs explain, there is much more to the positioning and supportive roles of pericytes than passive maintenance of the vessel wall.

5.8.1 PDGF-B and Pericyte Recruitment

Mural cells become associated with the forming vessels at later stages of development, which has led to the suggestion that the endothelial cells may govern vessel layer acquisition [88]. Accumulated evidence shows that PDGF-B plays a critical role in the recruitment of pericytes to newly formed vessels. In the developing mouse embryo, PDGF-B is produced by the sprouting endothelium, whereas PDGFR-b is present on developing pericytes [32, 85, 89], suggesting a paracrine mode of interaction between the two cell types. Targeted disruption of PDGF-B or PDGFR-b genes both lead to virtually identical phenotypes: perinatal lethality due to widespread microvascular leakage and hemorrhage [90, 91] caused by a severe deficit in pericytes [85]. Moreover, endothelium-specific PDGF-B knockout leads to pericyte deficiency [92]. In support of this theory, it has been demonstrated in an in vitro co-culture system that proliferating endothelial cells secrete PDGF-B, which acts as a chemoattractant and mitogen for undifferentiated mural cell precursors and induces a shape change from polygonal to spindle-shaped, reminiscent of smooth muscle cells in culture [93, 94]. It is well documented that pericytes become associated with endothelial cells while the vasculature is forming [78]. In fact, pericytes in culture have a select affinity for the endothelia [95]. The migration of pericytes to capillary-like structures was observed to be faster than that of astrocytes and pericytes covered these structures more extensively than astrocytes. We completed this in vitro BBB system by adding astrocytes to these mixed cultures of endothelial cells and pericytes [96]. Under these tri-culture conditions, endothelial cells reorganized into capillary-like structures with closely associated pericytes (Fig. 5.1 e, f), which allowed a definition of the heterocellular interactions involved in BBB development. The assembly of heterocellular complexes seems to be an essential step in the process of vessel maturation, because functional differentiation of ce-

5.8 Angiogenesis and Stability

rebral pericytes (pAPN-expression) was first acquired when association with endothelial cells occurred [43] and only the presence of pericytes guaranteed stability of the astrocyte-induced capillary-like structures and prevented regression. Taken together, tri-culturing of endothelial cells, pericytes, and astrocytes provides a reliable in vitro model by which cerebral angiogenesis and the underlying well balanced mechanistic effects can be mimicked. This view is fostered mainly by data from retinal angiogenesis, suggesting that a newly formed vascular network remains immature and “plastic” until pericytes are recruited [97]. The presence of pericytes in angiogenic sprouts suggests that they may be functional in the angiogenic process [98]. While pericytes appear to be normally induced in the absence of PDGF-B or PDGFR-b, the subsequent co-migration and proliferation of the pericyte population is impaired [32]. Detailed analysis of the microvasculature in PDGF-B and PDGFR-b mutants shows that angiogenic sprouting, at least in certain locations such as in the embryonic brain, proceeds relatively normally in the absence of pericytes and leads to a normal number of branch points and a normal microvessel density in this tissue [79]. However, blood vessels devoid of pericytes exhibit several other defects, such as irregular diameter, increased permeability, and rupturing microaneurysms at late gestation. The temporal correlation between failure of pericyte recruitment and endothelial hyperplasia suggests that the lack of pericytes directly regulates endothelial cell number [79]. Taken together, the available evidence therefore strongly implies that the endothelial cell-derived PDGF-B signal controls pericyte recruitment to angiogenic vessels and that pericyte growth during vessel formation may be positively regulated by PDGF-B.

5.8.2 TGF-b 1 and Differentiation

The above data suggest that the nature of the microvessel may be altered as pericytes attach to maturing capillaries. Pericytes are suspected to be responsible for the arrest of vessel formation and the induction of vessel maturation. Some groups were able to demonstrate that pericytes are capable of rendering endothelial cells quiescent [36, 99]. This pericyte-induced endothelial cell growth arrest and inhibited migration are mediated via contact and activation of TGFb1 [100]. Both endothelial cells and pericytes appear to synthesize a latent or inactive form of TGF-b1 when grown separately, but the activation of the protein may require the presence of both cells and physical contact, as implied from in vitro studies [99, 101, 102]. It is the activated TGF-b1 that then inhibits endothelial cell proliferation and migration. Thus, TGF-b1 in part constitutes a pericytederived signal for the endothelial cells. This factor also inhibits the proliferation of pericytes in culture [103], like it does endothelia, and induces at least in part the expression of contractile properties in these cells [40, 94]. These findings are supported by observations of mice null for TGF-b1 [104] or its receptor TGF-b receptor type II (TGF-bR2) [105] and co-receptor endoglin

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[106] that display defective endothelial-mesenchymal interactions and show defects in vascular wall structures. Thus, pericytes do provide a negative regulation of endothelial growth through TGF-b1, which is actually activated through participation of the endothelial cell itself.

5.8.3 Ang-1 and Maturation

Somewhere in the remodeling process lies the contribution of the angiopoietins. The angiopoietins Ang-1 and Ang-2 have been identified as ligands of the endothelial receptor tyrosine kinase Tie-2 [107–109], which controls vascular remodeling and maturation more than induction of angiogenesis. The largely complementary phenotypes of Ang-1- or Tie-2-deficient mice [108, 110] and Ang-2-overexpressing mice [107] have led to an antagonistic model in which Ang-1 acts as a Tie-2-activating agonist and Ang-2 acts as a Tie-2-inhibiting antagonist. These mice exhibit aberrant vascular development, characterized by abnormal interactions between endothelial cells and their supporting cells. A clue to the role of the angiopoietins is provided by examination of the expression patterns of the angiopoietins and their receptor. All endothelial cells express Tie-2 during development and in adult tissues, while perivascular cells are the primary source of Ang-1 [108, 109]. That pericytes contribute to the production of Ang-1 has also been shown in vitro [81–83]. Recently, it was found that Ang-1, acting via the Tie-2 receptor on endothelial cells, is essential for the remodeling and stabilization of the embryonic vasculature [111]. The authors demonstrated that, whereas blocking the function of the PDGFR-b in the developing retinal vasculature led to mural cell-deficient vessels that were poorly remodeled and leaky, administration of recombinant modified Ang-1 restored the vascular structure of the larger vessels in the absence of the mural cells. Ang-1 is also known as an anti-permeability factor and prevents vascular leakage [112]. It has been reported that administration of Ang-1 reduces BBB leakage in the ischemic brain [113]. Ang-2 has been identified as a functional antagonist of Ang-1 that binds to Tie-2 without inducing signal transduction [107]. Ang-2 expression is restricted to endothelial cells at sites of vascular remodeling. This pattern of expression led to a hypothesis that Ang-1 is involved in vessel stabilization whereas Ang-2 is a destabilizing factor. The expression level of Ang-2 is induced under hypoxic conditions in brain, whereas Ang-1 expression is unchanged [114]. Ang-2 function is considered to be contextually determined by the presence of other cytokines, for example, Ang-2 has a pro-angiogenic effect in the presence of angiogenic factors such as VEGF and induces vessel regression in the absence of angiogenic cytokines [115]. All these observations clearly demonstrate a strong interdependence between the endothelial cell and its intimate partner (the pericyte). The tightly controlled

References

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5.9 Conclusion

In essence, cerebral pericytes are emerging as an important member of the cellular complex that constitutes the BBB. The pericytes are complex cells, with metabolic, signaling and mechanical roles to support the endothelial cells. Endothelial cell-pericyte relationships are better understood and appear to involve complex biological feedback mechanisms between endothelial cells and pericytes. Interactions between these two cell types are important for the maturation, remodeling, and maintenance of the vascular system via the secretion of growth factors or modulation of the extracellular matrix. An important role for pericytes in pathology, and neuropathology in particular, has been indicated in hypertension, diabetic microangiopathy, Alzheimer’s disease, multiple sclerosis, and CNS tumor formation [116–121]. Putting the pericyte functions in context with the requirements of the environment may be the key to understanding pericyte nature.

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6 Brain Macrophages: Enigmas and Conundrums Frederic Mercier, Sebastien Mambie, and Glenn I. Hatton

6.1 Introduction

Macrophages are cells from hematopoietic origin that migrate and reside in all organs and tissues throughout adulthood. The central nervous system (CNS) is not an exception and contains numerous macrophages in the meninges, choroid plexus, circumventricular organs, and in the whole CNS vasculature. Brain macrophages are described as sentinels responsible for the brain immune defense and for clearing cell and extracellular matrix debris inherent in the maintenance of neural tissue. A fascinating emerging possibility is that macrophages, which are efficient producers of extracellular matrix molecules and growth factors/cytokines, have an influence on, and perhaps regulate, neuronal and glial cell functions as well as the proliferation and differentiation of neural precursor cells and neural stem cells (NSCs) throughout adulthood. In addition, because they are highly mobile cells, macrophages can migrate to sites of physiological challenge or injury in order to more efficiently deliver signaling molecules and ultimately influence the morphological and functional plasticity of neural tissue. First, we describe the various types of macrophages present in the adult brain, commenting on their distribution and ultrastructure. Macrophages have been, and often still are, confused with other resident cell types: pericytes, dendritic cells, and microglial cells. We show here that macrophages of the brain ventricle walls ultrastructurally resemble adult neuroblasts. Distinguishing these cells from one another is not easy, based upon only ultrastructural and location criteria. This is made even more difficult by cell “specific” markers such as CD cell surface antigens that can be up- and down-regulated with maturation, differentiation, and eventually trans-differentiation (differentiation from a given cell type to another cell type). We review macrophage functions starting with the conventional roles attributed to these cells, such as macrophagic activity and immune defense. We focus on the striking diversity of active molecules produced by brain macrophages, including extracellular matrix molecules and growth factors, and then analyze how the molecules may interact in the basal lamina at the astrocytic/macroBlood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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phage interface to ultimately regulate neural functions. Finally, we advance our view of the emerging possibilities that are suggested by recent literature, including the trans-differentiation of macrophages into neural cells.

6.2 Different Types and Locations of Brain Macrophages

Macrophages of the central nervous system (CNS) have various morphologies in the meninges, perivasculature, ventricle walls, ventricle lumen, choroid plexus, and circumventricular organs. Due to their high degree of morphological plasticity and because they migrate throughout and between these different locations, macrophages display different morphologies, even within compartments. Macrophages capable of phagocytic activity are primarily located in the CNS extraparenchyma (meninges, brain perivasculature, stroma of the choroid plexus and circumventricular organs), but microglial cells also function as phagocytes and are at least partly replaced by populations of macrophages that enter the neural tissue proper (parenchyma) after crossing the basal lamina located at the parenchymal/extraparenchymal interface (Hickey and Kimura 1988; Ling 1979; Ling and Wong 1993; Vallieres and Sawchenko 2003). The CNS cell types that can be considered to be macrophagic include microglia, pericytes, perivascular macrophages (PVM, also termed Mato cells or fluorescent granular perithelial cells; Mato et al. 1980), dendritic cells, Kolmer cells, epiplexus and supraependymal cells (Ling 1979; Ling et al. 1998; Lu et al. 1993). All these cell types vary in morphology within a given tissue compartment, migrate from one compartment to another, do not express strict specific markers that may distinguish the cells from each other in a given CNS compartment, and convert from one type to another in the different compartments upon the influence of cytokines and growth factors. It is, thus, currently impossible to state whether the CNS macrophage-type cells constitute one cellular entity with various morphologies, diversity of marker-expression, and different functions, or are morphologically and functionally distinct cell types. Even though, in lineage, they are most likely related to macrophages and may act phagocytically, we will not consider microglia and pericytes to be macrophages. The frequent confusion of pericytes with PVMs can be resolved ultrastructurally on the basis of their respective locations in the blood vessel wall, i.e. in tunica media for pericytes and in tunica adventitia for PVM (Bechmann et al. 2001 a; Graeber et al. 1989). Because the media and adventitia of arteries and arterioles form sleeves that completely cover the endothelium, PVMs and pericytes/smooth muscle cells can be distinguished from each other in their respective layers (Fig. 6.1). To distinguish the two cell types along capillaries where the media and adventitia form incomplete layers is more difficult (Fig. 6.2 A, B). This is why the existence of a capillary adventitial layer that consists of PVM has not been recognized in the past. Figure 6.3 C shows a capillary with easily identifiable and ultrastructurally distinct pericyte and PVM. Smooth


Fig. 6.1 Ultrastructure of arteriolar perivascular macrophages (PVMs). PVMs are located in the outer layer of the vasculature (adventitia), between astrocytes (Ast) and the smooth muscle cells (SMC) forming the media. Endothelial cells (End) form the intima. Basal laminae (large arrow) separate PVMs from Ast and SMC. PVMs possess

large lysosomes (arrow), a developed Golgi apparatus (arrowheads), and clumped electron-dense heterochromatin (large arrowhead). The electron density of the PVM cell body is intermediate between electron-lucent astrocytes (ASM) and electron-dense smooth muscle cells (SMC) and endothelium (End). Neu: neuronal processes. Scale bar: 500 nm.

muscle cells and pericytes are always electron-dense (in the media: Figs. 6.1, 6.3 C and 6.5 B). Most PVMs show an electron-density that is intermediate between pericyte/smooth muscle cells and astrocytes (Fig. 6.1). Pial macrophages, however, which can be likened to PVMs, are often electron-dense (Fig. 6.3 A). The adventitia represents an extension of the pia at the blood vessel surface. It has been established that macrophages migrate from the pia to the adventitial layer during inflammation (McKeever and Ballantine 1978). To our knowledge, there are no data suggesting that pial macrophages change electron-density when reaching the adventitia. Thus, the electron-lucent and electron-dense macrophages may either represent two different cell types or one single cell type that changes its ultrastructural characteristics.

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Fig. 6.2 Perivascular macrophages (PVMs) in the brain vasculature. (A) Microglial cell (Mic) abutting a PVM that displays lysosomes (arrows). Unlike Mics, PVMs are on the vessel side of the basal laminae (arrowhead). The large arrow indicates the Mic interface outside of basal lamina. End: endothelial cell. (B) Mic, identified by its large lysosomes (arrow), abutting a capillary. Arrowhead indicates subendothelial basal lamina. (C) Venous PVMs are directly apposed to

the surface of endothelial cells (End). A basal lamina (arrow) separates the PVM from surrounding astrocytes (Ast). Arrowheads indicate lysosomes. (D) Arteriolar PVMs contain smooth muscle cells (SMC) separating endothelium from PVMs. Note the presence of numerous large lysosomes in the PVM. The basal lamina overlying this PVM is thick (white arrow) or elongates between Ast (black arrowhead). All scale bars: 2 lm.


Fig. 6.3 Meningeal and intervascular macrophages. (A) Pial macrophage (Mac) located just beneath the basal lamina (arrow) underlying the astrocyte (Ast) endfeet of the glia limitans. Scale bar: 2 lm. (B) Intervascular macrophage (IVM) between two capillaries (Cap) of the hypothalamus. Arrays of axons (between

arrowheads), which are separated from the IVM by thin astrocytic endfeet, are found along the IVM. A microglial cell (Mic) is closely associated with the IVM. Scale bar: 2 lm. (C) Cytoarchitectonics of a capillary, showing the intima (End: endothelial cells), the media (Per = pericyte) and the adventitia (Mac = macrophage). Scale bar: 1 lm.

6.2.1 Macrophage Structure and Ultrastructure 6.2.1.1 Perivascular Macrophages The tunica adventitia of brain blood vessels consists of pia-arachnoid tissue. Macrophages and fibroblasts are the primary cells encountered in this tissue, although the cell ratio varies from a high fibroblast density in the meninges to a high macrophage density along the brain vasculature. The arterial adventitia resembles the meninges proper, and possesses a Virchow-Robin space that is continuous with the sub-arachnoid space (Mercier, 2004; Mercier and Hatton,

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Fig. 6.4 Meningeal and PVM network. (A) Electromicrograph showing the PVM network (highlighted in light purple) along an arteriole (with a lumen diameter of 13 lm) in the caudate putamen/corpus callosum interface. The basal laminae separating PVMs from smooth muscle cells (SMC) and astrocytes (Ast) is highlighted in dark blue. End: endothelium. Scale bar: 2 lm. (B) Immunofluorescence micrograph showing a triple label for CD163 (visualizing PVMs, red), laminin (visualizing blood vessel basal laminae, green) and bisbenzimide (visualizing cell nuclei, blue).

PVMs (arrows) are seen on most blood vessels (BV). IG: indusium griseum. Scale bar: 20 lm. (C) The lamina affixa, a meningeal tissue located between the thalamus (Th) and hippocampus (Hip), contain numerous PVMs (red). The lamina affixa/neural tissue interface is delineated by basal laminae (green). Laminin: green; bisbenzimide: blue. Scale bar: 50 lm. (D) Typical bipolar PVM labeled with CD163. An arrow indicates the cell body. Scale bar: 10 lm. (E) PVMs labeled with CD163 (red). Bisbenzimide: blue. Scale bar: 10 lm.

2004). The arterial Virchow-Robin space disappears as arteriole lumens approach 15–20 lm in diameter. Most arterioles with a lumen diameter less than 15 lm have PVMs tightly apposed to both the basal lamina overlying the media and the sub-astrocytic basal lamina (Figs. 6.1, 6.2 C, D and 6.4 A). Contrary to common belief, brain capillaries, even those having a lumen diameter of 6–8 lm, possess an adventitia filled with PVMs (Figs. 6.2 A and 6.3 C).


Fig. 6.5 Perivascular macrophages (PVMs) express collagen fibers. (A) Aligned fibers of collagen with a diameter of 35–50 nm in the PVM cytoplasm (arrow) and the thick basal lamina overlying the PVM (arrowhead). The PVM contains characteristic large lysosomes (Lys). Ast: astrocyte. Scale bar: 500 nm.

(B) PVM located at the interface of two merging arterioles (Art). 35–50 nm collagen fibers are visible in both the PVM cytoplasm (arrowhead) and the superficial basal lamina (arrows). Ast = astrocyte; End = endothelial cell; Lys = lysosome; SMC = smooth muscle cell. Scale bar: 1 lm.

Figure 6.1 shows the ultrastructure of a typical macrophage in the tunica adventitia of a brain blood vessel. PVMs are located between astrocytes and smooth muscle cells in large blood vessels, or astrocytes and pericytes in capillaries. Because pericytes only partly cover the capillary endothelium surface (approximately 30%), capillary PVMs can also be found directly inserted between astrocytes and endothelial cells. There are no fundamental ultrastructural differences between venous (Fig. 6.2 C), arteriolar (Figs. 6.1 and 6.2 D) and capillary (Fig. 6.2 A) PVMs. The PVMs display large lysosomes, some of which have a diameter of more than 1 lm (Fig. 6.1, arrow). The cytoplasm of PVMs is often filled with densely packed organelles, including Golgi apparatus, mitochondria, rough reticulum endoplasmic, and characteristic clusters of free ribosomes

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Fig. 6.6 Macrophages of the choroid plexus (CP) and ventricle walls. (A) Bi-nucleated dendritic cell in the CP stroma showing numerous tortuous cell processes. The insets (4.0 ´, top; 2.5 ´, bottom) show areas indicated by arrows in A, showing that dendritic cells possess clusters of free ribosomes (arrowhead) and intermediate filaments (large arrow). (B) Schema depicting the migratory pathway of monocyte-derived macrophages from the CP to the ventricle wall. The locations of the ultramicrographs shown in the figure are reported in the schema as their respective letters.

(C) Bipolar epiplexus cell (Kolmer macrophage) in the lateral ventricle. Arrow: lysosome. Arrowhead: CP epithelium. (D) Bipolar subependymal macrophage identified by a lysosome (arrow) and surrounding electron-lucent bands (arrowheads). Black arrow: subependymal basal lamina (fractone). (E) Neuroblasts of the lateral ventricle wall display characteristics of migrating macrophages: clumps of electron-dense heterochromatin, narrow cytoplasm, and surrounding electron-lucent bands. Black arrow: fractone. All scale bars: 2 lm.


Fig. 6.7 Ultrastructural similarities between macrophages and pre-migrating neuroblasts. (A) Pre-migrating neuroblasts in the subependymal layer of the lateral ventricle are surrounded by electron-lucent lateral bands (black arrows). Scale bar: 2 lm. (B) 4 ´ magnified field of the neuroblast cytoplasm indicated by a white arrow in A, showing clusters of free ribosomes (arrows). Scale bar: 200 nm.

(C) Subependymal macrophage (Mac) identified by the presence of a large lysosome (small arrow). Arrowhead: fractone. Scale bar: 1 lm. (D) 10 ´ magnified field of the Mac process indicated by a large arrow in C, showing clusters of free ribosomes (arrows). Lys = lysosome. Arrowhead: fractone. Scale bar: 100 nm.

(Fig. 6.7 C). PVM nuclei are ovoid and display a characteristic electron-dense heterochromatin with indentations (Fig. 6.1, large arrowhead; Fig. 6.2). Like pericytes, PVMs are encircled by a continuous basal lamina (Fig. 6.1, large arrow; Fig. 6.2 A, arrowhead; Fig. 6.3 C, arrow), except where PVMs contact one another to form a network (Fig. 6.4 A; Mercier et al. 2002). PVMs are often coated with a very thick (Fig. 6.2 D, arrow) or complex-shaped (Fig. 6.2 D, arrowhead) basal lamina. PVMs also connect adjacent blood vessels as intervascular macrophages (Fig. 6.3 B; see also Mercier et al. 2003).

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6.2.1.2 Meningeal Macrophages Macrophages are abundant throughout the meninges, at the surface of the brain, and in the projections located between major brain structures, such as the lamina affixa between hippocampus and thalamus. Pial macrophages, which contact the basal lamina underlying the glial limitans, are electron-dense and possess few large lysosomes (Fig. 6.3 A). Arachnoid macrophages contact fibroblast processes to form a network (Mercier et al. 2002), but whether the arachnoid macrophages form a network on their own is difficult to assess by transmission electron microscopy without serial sectioning and three-dimensional reconstruction over a large tissue volume. Along large blood vessels of the brain, PVMs can be considered as meningeal macrophages, bathing with fibroblasts (Mercier, 2004) in the Virchow-Robin/subarachnoid space.

6.2.1.3 Dendritic Cells According to recent studies, rat meninges and choroid plexus stroma contain numerous dendritic cells that are distinguished from the ED2 (also termed CD163) macrophages by their expression of the cell surface antigens OX6 and OX62, markers of major histocompatibility complex (MHC) class II (McMenamin, 1999; McMenamin et al. 2003). Dendritic cells were previously characterized in the CNS but were reported as rare in normal rodents, only being present in substantial numbers after acute injury or experimental autoimmune encephalomyelitis (EAE; Matyszak and Perry 1996; Matyszak et al. 1997). Dendritic cells, which originate from bone marrow after maturation in lymphoid organs, are considered as “sentinels” of the immune system. Dendritic cells are antigenpresenting cells (APC), express MHC class II antigens, and initiate T-cell responses. Macrophages do not express MHC class II in the normal brain, but only in activated macrophage subpopulations (McMaster and Williams 1979). Dendritic cells form networks in the choroid plexus and meninges (McMenamin 1999) as they do in peripheral organs and tissues (Steinman 1991). Morphologically, dendritic cells are pleiomorphic, i.e. dendriform, displaying numerous processes. One dendritic cell of the choroid plexus stroma is shown in Fig. 6.6 A. The cell has an electron-dense heterochromatin that is typical of the macrophage family, with indentations. The numerous processes of variable diameter make this cell distinguishable from a regular macrophage. Although it is believed that dendritic cells and macrophages may originate from different circulating monocyte precursors, the cells can convert to one another (Ardavin 2003; Dauer et al. 2003). Dendritic cells intervene in inflammation (Fischer and Reichmann 2001; Matyszak and Perry 1996). However, because these cells have only recently been characterized in brain, their specific function in brain is unknown. Interestingly, dendritic cells exist in the hypophysis (Sato and Inoue 2000), where they intervene in endocrine regulation (Hoek et al. 1997).


6.2.1.4 Ventricular Macrophages Different cell populations reside or transit in the brain ventricles. Ventricular cells have been identified by their location at the surface of the choroid plexus (epiplexus cells, also termed Kolmer cells), or ependyma (supraependymal cells; Scott 1999). Intraventricular, free motile cells are also found along the entire ventricular neuroaxis (Ling 1976). Most of these cells are macrophages (Bleier et al. 1975, 1982; Ling 1979; Ling and Wong 1993, 1982; Lu et al. 1993), although macrophages often cluster at the ependymal surface with T-lymphocytes and immature neurons expressing gamma aminobutyric acid (GABA) and serotonin (Del Brio et al. 1992; Harandi et al. 1986; Hirunagi et al. 1989). It has been demonstrated that epiplexus macrophages, free motile intraventricular macrophages, and supraependymal macrophages are the same cells at different locations along a migratory pathway extending from the choroid plexus stroma to the ventricle wall (Ling 1979; Ling and Wong 1993). Using carbon (Indian ink) and rhodamine labeling techniques, the authors have shown that circulating monocytes first enter the choroid plexus stroma, where they mature into macrophages. Then, the macrophages cross the choroid epithelium, transit in the lumen of the lateral ventricle as Kolmer cells, attach to the ependymal surface as supraependymal cells, and infiltrate the ventricle wall. In neonate rodents at least, some of the infiltrated cells mature into microglia (Ling 1979; Ling and Wong 1993). Brain infiltration of monocytes and their maturation into macrophages have been recognized in adult animals (for reviews, see Hickey 2001; Ransohoff et al. 2003), using models of bone marrow radiation chimeras (Lassmann et al. 1993) and bone marrow grafts from enhanced green fluorescent protein (EGFP) mice (Vallieres and Sawchenko 2003). All data support the view that brain macrophages are constantly replaced by blood-derived cells throughout life. Most macrophages stay within the meninges, perivascular layer, and choroid plexus. However, in agreement with the theory of Rio-Hortega (1932), a fraction of these macrophages enter the neural compartment to replenish microglia. It is however not clear whether all microglia derive from macrophages or whether all microglia and macrophages derive from separate precursors (Walker 1999). It is also discussed, using ultrastructural similarity, that infiltrated ventricular macrophages may represent a source of neural stem cells or neural progenitor cells (further explained in a later section).

6.2.2 Immunotyping by Cell Surface Antigens

There is a considerable heterogeneity in the immunophenotype of brain macrophages, dendritic cells and microglia (Graeber et al. 1989). These cells express several cell-surface antigens termed CD, ED, or OX, which greatly vary among species, organs, location in the tissue, and the degree of physiological or pathological activation. The common confusion of macrophages, pericytes, and microglia (Guillemin and Brew 2004; Kida et al. 1993; Thomas 1999) has led to sev-

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eral mistakes. In addition, some markers thought to be specific for other cells have been later found in macrophages. For example, the lymphocyte markers CD4 and CD8 are expressed by healthy and diseased brain macrophages (Perry and Gordon 1987; Perry et al. 1987; Popovich et al. 2003). CD14 is present in both macrophages and microglia (Beschomer et al. 2002); and numerous markers have been characterized in diseased brain (AIDS, auto-immune encephalomyelitis, Alzheimer’s disease, Parkinson’s disease). Also, it is important to remember that macrophages are scavenger cells that phagocytose necrotic or apoptotic cells. By phagocytosis or pinocytosis, macrophages can absorb cell markers of foreign origin. It has been shown, for example, that macrophages can absorb myeloperoxidase activity from ingested neutrophils (Graeber et al. 1990; Wendling et al. 1991). However, in the rat, CD163 is a reliable marker of PVMs (Fig. 6.4 B, D, E) and meningeal macrophages (Fig. 6.4 C; Angelov et al. 1996).

6.2.3 Macrophages Contact Basal Laminae

All brain macrophages, except those located in the ventricle lumen, contact a basal lamina by their cell body or processes. PVMs are totally enclosed by basal laminae (Fig. 6.1). Macrophages of the choroid plexus stroma contact the basal lamina underlying the epithelial cells; and those of the ventricle walls contact the fractones, the specialized subependymal basal laminae (Mercier et al. 2003). Pial macrophages directly contact the basal lamina interfacing the pia and glia limitans (Fig. 6.3 A). Macrophages of the arachnoid cell barrier layer (at the interface between dura and arachnoid) also contact a basal lamina (Vandenabeele et al. 1996). In addition, the punctate basal laminae that exist throughout the arachnoid (Mercier 2004; Mercier and Hatton 2001) often contact macrophages or fibroblasts. It is likely that macrophages participate in the synthesis and deposition of extracellular matrix (ECM) molecules composing the basal lamina. Collagens IV, XV, and XVIII and laminins b-1 and c-1, which are the principal basal lamina components, are produced by fibroblasts, but the absence of fibroblasts along capillaries and in the subependymal layer (SEL) of the entire ventricular system suggests that basal laminae are there produced by other cells. The characterization of collagen fibrils within and at the surface of PVMs strongly suggests that PVMs produce collagens (Fig. 6.5). It is known that macrophages are the primary producers of the metalloproteinases MMP-2 and MMP-9, which are the principal enzymes involved in the degradation of basal laminae. Macrophages also produce heparan sulfate proteoglycans (HSPG) present in basal laminae, such as perlecan, a crucial binder and activator of heparin-binding growth factors. The heparin-binding growth factors are among the most powerful signaling molecules, intervening in cell proliferation, differentiation, and migration. But these factors are usually inactive or weakly active when free in the extracellular space and need to be activated and pre-


sented by the HSPG of the basal laminae to reach their full potential. Thus, the production of HSPG by macrophages and the interactions of these HSPG with growth factors in front of macrophages within basal laminae are likely very important for the control of cell renewal, cell differentiation or maturation, and any sort of morphological plasticity.

6.2.4 Network of Macrophages Through the Brain

PVMs contact each other via their elongated processes to form a network all along the brain vasculature. The PVM network has been previously characterized both at the light microscopic (Cuff et al. 1996) and ultrastructural levels (Mercier et al. 2002). Although the PVM network has been primarily described in arterioles, both venous (Fig. 6.2 C) and capillary (Fig. 6.3 C), PVMs belong to the macrophage perivascular network. The ultramicrograph in Fig. 6.4 A shows the PVM network in an arteriole of the lateral ventricle wall. The PVM network can also be visualized by labeling PVMs with the specific cell surface antigen CD163 (Fig. 6.4 B–E), although it is not established that CD163 stains all PVMs. Immunolabeling for CD163 visualizes both the cell body and process surfaces of PVMs (Fig. 6.4 D, E), but the process terminals either do not express CD163 or are not detected with the sensitivity of the antibody in our immunochemistry protocol. Thus, the PVM visualized by CD163 immunolabeling appears as dashed lines along the vasculature (Fig. 6.4 B). Macrophages present in meningeal infoldings with the brain, like those of the lamina affixa, are also labeled with CD163 (Fig. 6.4 C). The PVMs of the superficial meninges also form a network that can be visualized by transmission electron microscopy and immunolabeled with CD163 (not shown). PVMs and meningeal macrophages are constantly repopulated from bone marrow (Hickey et al. 1992) with a turnover rate of 3–4 weeks (Bechmann et al. 2001 a). These cells are known to be efficient APCs, even in comparison with microglial cells (Fabriek 2005 a; Hickey and Kimura 1988), and to participate in the brain immune defense and inflammation (Schiltz and Sawchenko 2003; Williams et al. 2001). Although PVM and meningeal macrophages express numerous cytokines, growth factors, and extracellular matrix molecules, their specific role(s) remain to be determined. The PVM network (Cuff et al. 1996) runs along the adventitial layer of the vasculature into the CSF compartment (Virchow-Robin space) that is continuous with the subarachnoid space of the meninges. In the meninges, macrophages also form a network that is interwined with a fibroblast network connected by functional gap junctions (Mercier and Hatton 2001; Spray et al. 1991). The gap junction protein Connexin (Cx)43 appears to be a key player in the development of progenitor cells and is expressed by macrophages, neutrophils and mast cells (Oviedo-Orta and Evans,2004). Moreover, gap junctional communication has been found between particular macrophages (foam cells of athero-

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sclerotic lesions; Polacek et al. 1993). Thus, it is also possible that brain macrophages communicate to each other via gap junctions throughout the CNS. Interestingly, macrophages inhibit gap junctional communication and downregulate Cx43 expression in cultured astrocytes (Rouach et al. 2002).

6.3 Migration of Brain Macrophages

Macrophages are highly mobile cells that can move throughout the meninges, perivascular layer (Virchow-Robin space), and ventricles. They are particularly mobile after brain injury or bacterial infection, converging at the injury site, where they secrete pro-inflammatory cytokines and ultimately induce an inflammation. Brain macrophages are further assisted in this task by monocytes and lymphocytes infiltrating from the circulation. Inflammation leads to a serious disturbance of the cytokine and growth factor microenvironment (see Chapter 11), which then may lead to repair or partial repair, but often induces astrogliosis and neuronal death. Meningeal macrophages can also cross the glia limitans, as shown in the suproptic nucleus after dehydration (Mercier 2004). The purpose of this migration into the brain parenchyma is unexplained but may be related to morphological changes associated with neuroendocrine regulation (Beagley and Hatton 1992; Hatton 1997).

6.4 Fast Renewal of Brain Macrophages

Macrophages have a short life span, being replaced every 3–5 weeks. It has been shown that macrophages are replenished from circulating monocytes, themselves of bone marrow origin (Hess et al. 2004; Hickey and Kimura 1988; Hickey et al. 1992). Monocytes are attracted by chemotaxy to cross the vessel wall. Macrophage chemo-attracting protein-1 (MCP-1) is one of the principal chemokines implicated in this process, termed extravasation. MCP-1 also induces migration of neural stem cells (Widera et al. 2004). Once past the endothelium and eventual smooth muscle cell layer, the monocytes mature into PVMs or dendritic cells. Circulating dendritic precursors also exist; and the conversion of PVMs into dendritic cells also occurs in the perivascular layer. Some PVMs and choroid plexus macrophages also continue their voyage to reach the brain parenchyma, where they mature into microglia. However, both macrophages and microglial cells are also capable of self-renewal in their respective compartments and thus they are replenished only partly by infiltration of new monocytes from the circulation. However, microglia replace themselves at a much slower rate than macrophages. The entry of monocytes in the adult brain occurs mainly in the choroid plexus and arterioles in both brain and meninges (Ransohoff et al. 2003). Because the macrophages have a short life, one may

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wonder whether the molecules and debris absorbed by phagocytosis are released in the neural microenvironment. Experiments based on the injection of carbon particles (Indian Ink) into brain have demonstrated that carbon particles are kept inside brain macrophages for at least 2 years, a result that is not consistent with the short life of macrophages (Hickey 2001). A possible explanation is that carbon particles can be successively re-absorbed by new macrophages after the death of the old ones.

6.5 Functions 6.5.1 Known Functions of Brain Macrophages

PVMs and other macrophages are primary soldiers in the brain’s immune defenses. These cells use conventional weapons such as phagocytic activity, which consists in the absorption and digestion of pathogens and cellular debris. Macrophages also act as APCs, cooperating with T lymphocytes to direct the specific immune response. Macrophages produce less conventional weapons that directly benefit neural cell functions, secreting signaling molecules such as cytokines, growth factors, and ECMs, the last of which influence growth factor activity in the neural environment. The resident CNS macrophages are not the only soldiers available to defend the brain after a challenge. As alluded to earlier, by a mechanism termed extravasation, which involves chemokines produced by vascular cells and resident PVMs, circulating monocytes enter the brain and accumulate at the sites of injury or physiological challenge. In the infiltrating pathways (vasculature, meninges, choroid plexus), the monocytes mature into macrophages, expand their lysosomal arsenal, and eventually become dendritic cells that produce MHC class II molecules, participating in the immune defense (Ransohoff et al. 2003). Ironically, infiltrating circulating monocytes, if infected by viruses, also serve as carriers for pathogens, as “trojan horses” that are welcomed by the brain, but they quickly betray their host after crossing the blood-brain barrier (BBB). Upon crossing the BBB, the HIV-infected macrophages insinuate through the neural tissue, a disastrous situation that leads to the destruction of neural cells and can ultimately trigger dementia (Williams et al. 2001). The infiltrating macrophages also participate in the progression of most neurodegenerative processes, accentuating the inappropriate composition of the altered neural environment. However, under physiological conditions, the infiltrating macrophages are beneficial to the CNS.

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6.5.1.1 Phagocytosis PVMs, other brain macrophages, and (to a lesser extent) microglia, via their numerous and sizeable lysosomes, have a large capacity for engulfing and digesting cellular, matrix, and pathogen debris. Macrophages phagocyte neural cells, vascular cells, and inflammatory cells that comprise macrophages themselves (Chan et al. 2003). To perform their phagocytic activity against pathogens, macrophages express receptors directed against bacterial and fungal components, such as peptidoglycan, teichoic acid, lipopolysaccharide (LPS), mannose (Galea et al. 2005), flagelin, and glucans, or viral components such as doublestranded RNA. Low pH, hydrolases, and peroxidases, and a lack of appropriate nutrients prevent the ingested parasitic organisms from replicating within the macrophage lysosomes. The phagocytic activity and release of hydrolases is linked with the immune function of macrophages (Cardella et al. 1974).

6.5.1.2 Immune Function In all organs and tissues, including the brain, macrophages and dendritic cells act as APCs. Antigens that have been previously absorbed and digested are presented to the “attention” of the T lymphocytes, which then initiate the immune response. Macrophages produce growth factors and cytokines that create or enhance immune responses in collaboration with other immune cells. Among these growth factors, macrophage colony stimulating factor (M-CSF) and granulocyte macrophage (GM)-CSF induce chemotaxis, phagocytosis, proliferation, differentiation, and maturation of hematopoietic stem cells and leucocyte progenitors (Cairo et al. 1990). Interestingly, GM-CSF also participates in the differentiation of neural progenitor cells (Kim et al. 2004) and in brain inflammation (Franzen et al. 2004). Macrophages also produce vascular endothelial growth factor (VEGF), which increases vascular permeability and contributes to the afflux of lymphocytes and monocytes (Lee et al. 2002). The presence of PVMs along the vascular adventitia of most blood vessels (Mercier et al. 2002) and their mobility (Cuff et al. 1996) make these cells particularly suitable for intervening rapidly at every level of the brain and initiating an appropriate immune response (Bechmann et al. 2001 b; Mato et al. 1996). Migrating intraventricular macrophages also participate in the immune and inflammatory response of the neural tissue (Maxwell and McGadey 1988). Evidence that the brain microenvironment and macrophages/microglia intervene in the brain immune surveillance and defense has been intensively reviewed. The reader is invited to read the excellent reviews of Becher et al. (2000), Fabriek et al. (2005 b), and Thomas (1999).

6.5.1.3 Production of Growth Factors, Cytokines, and Chemokines Although not the only cells, macrophages produce several growth factors, including basic fibroblast growth factor (FGF-2), transforming growth factor-beta1 (TGF-b1), and tumor necrosis factor a (TNFa; Mato et al. 1998). In brain, FGF-2

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is primarily produced by endothelial cells, ependymal cells, macrophages (Frautschy et al. 1991; Grotte et al. 2001), and choroid plexus cells (Johanson et al. 2001). Monocytes and macrophages also produce hormones that are identical to neuronally produced hormones, for example corticotropin releasing hormone (CRH), which locally activates the inflammatory response (De Souza, 1995). Macrophages produce inflammatory and anti-inflammatory cytokines, such as interleukin-1b (Angelov et al. 1998 a, b; Bauer et al. 1993). It was previously thought that circulating cytokines have their effects on the brain only via circumventricular organs, which have a leaky, if any, BBB. It has been demonstrated that circulating cytokines induce the production of the same cytokines by PVMs, pia-arachnoid macrophages, and microglia. Because all these cells are inside the BBB, the cytokines released are directly accessible to glia and neurons. Cytokines have strong effects on brain function, generating fever, behavior changes, stress responses, and multiple effects due to their influence on the neuroendocrine system (for a review, see Mercier and Hatton 2004). Cytokines released by macrophages are also capable of inducing neuronal death by necrosis or apoptosis, astrocytosis, and the proliferation and differentiation of neural progenitor cells and NSCs. Brain macrophages are themselves activated by other immune cells, such as lymphocytes, which constantly co-enter the brain with monocytes during physiological (discussed in a later section) and pathological conditions. For example, lymphocytes produce growth hormone, which activates macrophages. In addition, brain macrophages attract circulating monocytes by producing the chemokine monocyte chemotactic protein-1 (MCP-1; Calvo et al. 1998). This is used by macrophages that have already been recruited into an inflammation site to further recruit other macrophages. In the neurohypophysis, macrophages also produce inducible nitric oxide synthase (iNOS; Gajkowska et al. 1999). Nitric oxide is known to serve as a neuromediator in brain.

6.5.1.4 Production and Degradation of the Extracellular Matrix Macrophages are an important source of ECM molecules. Macrophages produce matrix metalloproteinase (MMP)-2 and MMP-9, two collagenases that are involved in basal lamina and ECM degradation. Macrophages also produce urokinase-type plasminogen activator (uPA), an enzyme used to clear apoptotic and necrotic cells, and cyclo-oxygenase-2 (COX-2; Elmquist et al. 1997). Macrophages also express iNOS (Mato et al. 1998). Nitric oxide, which is also supplied by perivascular nerve fibers, is released in the perivasculature, particularly abundantly in the arteries of the Circle of Willis, where it may intervene as a co-neurotransmitter (Edvinsson et al. 2001).

6.5.1.5 Repair After Injury Until quite recently, it was thought that the CNS lacked any capability of self-repair after injury. Recent evidence, involving cell therapy with ensheathing glia, demonstrates the opposite: self-repair of the transected spinal cord in the adult rat (Ra-

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mon-Cueto 1998, 2000). In this model, anatomical recovery (axonal reconnection through 4 mm of missing tissue) preceded by several weeks the recovery of sensorimotor functions. Although the mechanisms of repair induced by injected ensheathing glia are not understood, it is likely that these cells release molecules that counterbalance an inhibitory spinal cord microenvironment resulting from the activity of local growth factors, cytokines, and ECM molecules (Becker and Becker 2002; Coumans et al. 2001; Krekoski et al. 2001). Jones et al. (2003) have shown that basal lamina molecules inhibit axonal growth. It is thought that the inflammation produced by fibroblasts and macrophages attracted by chemotaxis in the wounded area, and the associated glial scar, inhibit neural tissue repair. However, basal laminae also promote cytogenesis and repair throughout all organs and tissues in adulthood (Bunge 1987; Fujimoto et al. 1997; Gospodarowicz et al. 1981). It was recently demonstrated that macrophages produce trophic factors with both positive and negative effects on optic nerve regeneration (Yin et al. 2003). Macrophages also produce and release growth factors and ECM molecules promoting cytogenesis and cell differentiation (discussed in a later section). It is likely that connective tissue fibroblasts and macrophages and their adjacent basal laminae govern tissue plasticity everywhere in the body and thus both promote and inhibit cell proliferation, differentiation, and migration to an extent that is appropriate for the function of the tissue. It was recently shown that dendritic cells promote the repair of injured spinal cord (Mikami et al. 2004). The authors demonstrated that dendritic cells strongly induce the proliferation and survival of neural progenitor cell and neural stem cell activity.

6.5.2 Potential Functions of Brain Macrophages 6.5.2.1 Interactions with Meningeal/Vascular Cells, Neurons, and Astrocytes Macrophages are strategically located at the interface between astrocytes and the connective tissue/CSF compartment. The CSF potentially carries information from all over the brain, and from peripheral tissues if the fluid that contacts macrophages is outside of the BBB (for example in the stroma of the choroid plexus). All macrophages have access to CSF, carriers of signaling molecules, and the basal lamina permits exchange of information with the other cell types. SEL macrophages/microglia also have access to CSF information by interstitial clefts that connect the ventricles to the extracellular space bathing the SEL cells (Brightman 1965, 2002). The astrocyte/macrophage contact occurs directly in the SEL or indirectly via the basal lamina located between the two cell types, at the pia/glia limitans interface or at the blood vessel surface. Thus, macrophages and astrocytes can exchange information via signaling molecules that may be activated in the basal laminae. Eventually, macrophages can enter the neural tissue to further influence astrocytes and neurons. Migration of meningeal macrophages was observed in the supraoptic nucleus after dehydration (Mercier 2004). In the supraoptic nucleus, both macrophages lo-

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cated in pia and the infiltrated macrophages may produce growth factors and cytokines to signal astrocytes (and neurons?) of a homeostatic change (osmolarity change) and initiate morphological and functional changes in the neural tissue that will lead to osmotic regulation. It has been demonstrated that following dehydration, astrocytes retract processes from between adjacent neurosecretory neurons, which in turn have more opportunity to contact each other and coordinate the release of the neurohormones vasopressin and oxytocin (Hatton 2004). Another possible example of the influence of macrophages on neural tissue is given in the next section.

6.5.2.2 Do Macrophages Govern the Neural Stem Cell Niche in Adulthood? Adult Neurogenesis Neurons and glia (both neural cells) are continuously generated in the brain of adult vertebrates (Altman 1962 a, b; Ekstrom et al. 2001; Goldman and Nottebohm 1983; Lois and Alvarez-Buylla 1993; Luskin 1993). In mammals, the vast majority of neurogenesis occurs in the SEL of the lateral ventricles (Doetsch et al. 1997; Eriksson et al. 1998; Lois and Alvarez-Buylla, 1993) and dentate gyrus (Altman and Das 1965; Cameron et al. 1993; Seri et al. 2004). In the SEL of the lateral ventricle, the new neural cells are produced from self-renewing and pluripotent (with potential to produce neurons, astrocytes, oligodendrocytes) NSCs. These can be grown in culture as free-floating spherical aggregates, termed neurospheres, which contain new NSCs, neurons, astrocytes, and oligodendrocytes (Reynolds and Weiss 1992; Weiss et al. 1996). NSCs have been found throughout the entire ventricular system in adult mammals, but for unknown reasons, the SEL of the lateral ventricles is more mitotically active and supports the production of both neurons and glia, while the SEL of the other ventricles and spinal cord shows poor mitotic activity and favors gliogenesis. NSCs are not clearly identified in vivo. Controversial data suggest that NSCs may be ependymocytes (Johansson et al. 1999), astrocytes (Doetsch et al. 1999), or radial glial cells originating from embryonic neuroepithelium (Mercle et al. 2004). Moreover, sufficient evidence has not been provided that neuroblasts, which are ultrastructurally identifiable unipotent neural precursor cells, derive from NSCs, although the prevalent theory suggests such a lineage (AlvarezBuylla et al. 2001; Doetsch et al. 1999). The Neurogenic Niche The concept of “microenvironment” or “niche” regulating NSCs proliferation and differentiation in the SEL was recently proposed (see Chapter 3). In support of this concept, it was shown that the fate of a precursor or stem cell depends on the niche in which it is housed. The induction mechanisms from the niche may even overrule the genetic cell commitment. For example, hippocampus-derived neuroblasts transplanted into the olfactory bulb differentiate into olfactory neurons and not hippocampus-type neurons (Suhonen et al. 1996). NSCs can even trans-differentiate into non-neural cells, for example into hematopoietic cells, after transplantation in bone marrow

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(Bjornson et al. 1999). However, the exact components, as well as the mechanisms orchestrating the niche and ultimately determining cell fate, are poorly characterized. It is known that neurogenesis is regulated by growth factors, cell adhesion molecules (Amoureux et al. 2000), and hormones (Nordeen and Nordeen 1989; Rasika et al. 1994; Schingo et al. 2003). From a number of previous studies, it is apparent that several growth factors are involved in determining the fate of NSCs. These include heparin-binding growth factors such as FGF-2 (Gritti et al. 1996; Kuhn et al. 1997), amphiregulin (Falk and Frisen 2002), heparin-binding growth associated molecule (HB-GAM, also termed pleiotrophin; Hienola et al. 2004), TGFb-1 (Zhang et al. 1997), interferon gamma (IFN)c (Wong et al. 2004), interleukin (IL)-6 (Vallieres et al. 2002), bone morphogenetic proteins (Lim et al. 2000), and heparin-binding epidermal growth factor (HB-EGF; Jin et al. 2002). After their release from the cells and diffusion in the extracellular space, the heparin-binding growth factors are captured by HSPG, which functions as a dynamic local storage and modulator of growth factor activity. HSPG present growth factors to cell-surface receptors, initiating a triple interaction that is required for triggering an intracellular protein kinase cascade. Alternatively, the HSPG/growth factor complex is internalized by endocytosis and reaches the cell nucleus, where the growth factor serves as a transcription factor for cell proliferation (Allen and Rapraeger 2003; Chipperfield et al. 2002; Reiland and Rapraeger 1993). Despite the accumulated evidence for a crucial role of HSPG in morphogenesis, stem cell activation, and hematopoiesis during development (Gilbert 1994, 2001; Gordon et al. 1987; Roberts et al. 1988), there is little information as to the role of HSPG in adult NSC niches. It has been shown that embryonic neuroepithelial cells secrete HSPG that bind FGF-2 to direct their own cellular response (Brickman et al. 1995) and that laminin and fibronectin are permissive substrates for outgrowth and migration velocity of neurosphere cells in vitro (Kearns et al. 2003). We recently characterized subependymal basal laminae (fractones; see Figs. 6.6 D, E and 6.7 C) in direct contact with neural progenitor cells, ependymocytes, and astrocytes, in the ventricle walls (Mercier et al. 2002, 2003). Macrophages/ microglia are also very often associated with fractones, a location that may reflect the role of these cells in ECM molecule production/degradation and the structural/functional dynamics of fractones. Fractones consist of collagen-IV, laminin b-1 and c-1, nidogen, and the HSPG perlecan (Mercier, unpublished observations). Perlecan is known as the primary HSPG that binds the powerful neurogenic factor FGF-2 (Reiland and Rapraeger 1993). Moreover, we have demonstrated that biotinylated FGF-2 injected into the lateral ventricle of the adult rat brain exclusively binds to fractones. These observations suggest that fractones are involved in the biological activation of FGF-2 and potentially the neurogenic effects of this signaling molecule during adulthood. Macrophages in the Neurogenic Niche We have ultrastructurally identified macrophages/microglia all along the walls of the lateral and third ventricle (Mercier et al. 2002, 2003). In the most neurogenic zone, i.e. in the SEL at the

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caudate putamen/corpus callosum interface, we found large PVMs, containing numerous lysosomes (Figs. 6.1 and 6.2 D). These PVMs were associated with extensions of basal laminae and were directly connected to fractones via fractone stems (Mercier et al. 2002). In addition, immature macrophages/microglia bearing short processes were systematically associated with fractones (Figs. 6.6 D and 6.7 C) along the SEL of the third and lateral ventricles (Mercier et al. 2003). Three observations support the possibility that the immature macrophages/microglia associated with fractones in the NSCs niche are intraventricular macrophages (supraependymal/Kolmer cells) that infiltrated the ventricle wall. First, intraventricular macrophages derive from circulating monocytes and then transit throughout the choroid plexus before reaching the ependymal surface (Ling and Wong 1993). Second, the ultrastructure of intraventricular macrophages (Fig. 6.6 C) and SEL immature microglia (Figs. 6.6 D and 6.7 C) is identical. Common ultrastructural characteristics include dark and indented heterochromatin, dark and narrow cytoplasm containing some large lysosomes and numerous clusters of free ribosomes (Fig. 6.7 D), and large short processes (usually the cells are bipolar). Third, Ling and Wong (1993) have shown in neonatal rats that monocyte-derived macrophages infiltrate the SEL of the lateral ventricle, where maturation of these cells into microglia takes place. The last point is crucial in describing the entrance of bone marrow-derived cells into the choroid plexus and then the lateral ventricle wall. Because unidentified bone marrow cells migrating into the brain can trans-differentiate into glial cells and neurons in adulthood (Cogle et al. 2004; Eglitis and Mezey 1997; Mezey et al. 2000), it is possible that the pathway of monocytes-derived macrophages described by Ling and colleagues in neonates exists in adults, and that these macrophages become neural cells after infiltration in the lateral ventricle wall, known as the neurogenic niche in adulthood. Role of Macrophages in the Adult Neurogenic Niche No information is currently available on the specific roles of macrophages in the adult neurogenic niche. Macrophages are an abundant source of growth factors, cytokines and ECM molecules in peripheral organs and in brain after inflammation, but a current paucity of literature on the topic makes it difficult to evaluate whether this is the case in the normal brain. In addition, because the different cell types of the macrophage families are often confused, the identity of the cells producing signaling molecules is often unclear. During inflammatory responses and in the diseased brain, brain macrophages produce leukemia inhibiting factor (Getchell et al. 2002), TGF-b1 (Kiefer et al. 1998), HSPG (Jones et al. 2000), FGF-2 (Clasper et al. 1999), and unidentified toxic factors (Bragg et al. 2002). Macrophages/microglia express perlecan in response to hippocampal b-amyloid infusion (Miller et al. 1997). The circulating precursors of macrophages (monocytes) express HB-EGF (Reape et al. 1997), versican, and perlecan (Makatsori et al. 2003). Macrophages also express the anti-angiogenic thrombospondin (Di Pietro and Polverini 1993). Particularly interesting is the fact that PVMs identified by CD163 or CD14, both in vitro and in vivo, express the cell sur-

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face antigen CD44 (Jones et al. 2000), a HSPG that binds the powerful neurogenic factors FGF-2 and HB-EGF (Bennett et al. 1995). Jones et al. (2000) further demonstrated that CD44 are intermediate affinity receptors for FGF-2 and HB-EGF, present growth factors to their specific receptors on adjacent cells, and upregulate the activity of these growth factors. These authors suggested that macrophages regulate the bioavailability of heparin-binding growth factors to trigger cell proliferation and differentiation at the appropriate time and location. Interestingly, Jones et al. (2000) also demonstrated in vitro that CD44 HSPG expression increases more than five-fold when differentiation from monocytes to macrophages is induced. If monocyte-derived macrophages of the CNS behave similarly, these cells may express HSPG after extravasation, once in contact with subpial, perivascular basal laminae, or fractones in the case of choroid plexus-derived supraependymal cells. Interestingly, Jonakait et al. (2000) demonstrated that macrophages express molecules that synergize with nerve growth factor and influence differentiation of progenitor cells. Based on our findings that: (1) SEL macrophages are systematically associated with fractones (Mercier et al. 2002, 2003), (2) perlecan is specifically found in fractones, and (3) heparin-binding neurogenic factors, including FGF-2, are specifically sequestered into fractones after intracerebrovascular injection (Mercier et al. unpublished observations), we propose that the mechanisms of HSPG/growth factor interactions are the means by which macrophages operate in neurogenesis during adulthood. As crucial sites of ECM/growth factor interactions, fractones may activate the neurogenic growth factors and initiate their presentation to the receptors of abutting NSCs and neural progenitor cells, or participate in the mechanisms of internalization of the growth factors into the cells for activation of mitosis and differentiation. Similar mechanisms have been previously described for the mitotic action of FGF-2, although not in the context of neural stem cell biology in adulthood (Allen and Rapraeger 2003; Reiland and Rapraeger, 1993). Can Macrophages Trans-Differentiate into Neural Cells or Become Neural Stem Cells? It has been clearly demonstrated with both carbon and rhodamine labeling techniques that circulating monocytes enter the choroid plexus stroma, where they become macrophages. As macrophages, the cells cross the choroid epithelium, transit in the lumen of the lateral ventricle as Kolmer cells, attach to the ependymal surface as supraependymal cells, and infiltrate the lateral ventricle wall (Fig. 6.6 B). Some of these infiltrating cells mature into microglia in neonatal rodents (Ling 1979; Ling and Wong 1993). The choroid plexus-ventricle wall migration pathway has also been recognized in adult animals (for reviews, see Hickey 2001; Ransohoff et al. 2003), using models of bone marrow radiation chimeras (Lassmann et al. 1993) and bone marrow grafts from EGFP mice (Vallieres and Sawchenko 2003). All available data support the view that cell populations within the meninges, choroid plexus, as well as perivascular macrophages and microglia, are replaced by blood-derived cells throughout life. None of these studies support the view that bone marrow-derived cells trans-differentiate into neural cells after infiltration in the SEL, but the studies of Cogle et al. (2004)

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and Mezey et al. (2000) appear to suggest just this. Indeed, several studies demonstrate that trans-differentiation from a cell type to another cell type is possible (Bjornson et al. 1999; Kondo et al. 2000). Our hypothesis is that supraependymal macrophages originating from circulating monocytes and transiting via the choroid plexus as Kolmer cells naturally infiltrate the lateral ventricle – the neurogenic niche – and have the ability to trans-differentiate into neural cells, or to acquire multipotency, i.e. to become NSCs. Supporting the possibility that monocytes potentially trans-differentiate into other cells after extravasation, it has been demonstrated in vitro that monocytes can transform into endotheliallike cells in the presence of angiogenic factors (Fernandez-Pujol et al. 2000; Haveman et al. 2003). Second, the neuroblasts present in the adult neurogenic niche, termed “A” cells in the nomenclature adopted by Doetsch et al. (1997), possess numerous ultrastructural features in common with macrophages. Figure 6.7 A shows typical clusters of A cells present in the SEL at the caudate putamen/corpus callosum interface. The A cells display electron-lucent bands at their periphery, have a dense heterochromatin with some clustering, narrow perinuclear cytoplasm, and one or two large and short processes filled with numerous organelles, including a high density of clustered free ribosomes (Fig. 6.7 B). Macrophages present in the SEL of the ventricular system possess similar ultrastructural characteristics, displaying one or two large processes (Figs. 6.6 C and 6.7 D), a dark heterochromatin, and the characteristic electronlucent lateral bands (Figs. 6.6 D and 6.7 C) and dense clusters of free ribosomes encountered in no other cell type in brain, besides neuroblasts and cells of the macrophage family. Interestingly, dendritic cells in the choroid plexus stroma, as well as epiplexus cells also display these numerous clusters of free ribosomes in their processes (Fig. 6.6 A, insets). The only noticeable difference between cells of the macrophage family and neuroblasts is the presence of few large lysosomes in macrophages (Figs. 6.6 C, D and 6.7 C, D). The intriguing perspective is that macrophages may enter the neurogenic niche via the lateral ventricle lumen and trans-differentiate into neuroblasts, or even acquire neural stem cell properties upon influence of the neurogenic niche microenvironment. It is possible that macrophages lose their lysosomes upon trans-differentiation. In response to injury, microglia can express the hematopoietic stem cell marker CD34 (Labedy et al. 2005). That the proteoglycan NG2, a marker of oligodendrocyte precursor cells, is expressed by macrophages (Bu et al. 2001) is also going in the same direction.

6.5.2.3 Role of Macrophages in CNS Angiogenesis Blood vessels constantly reconstitute in the adult CNS. New endothelial cells, smooth muscle cells, pericytes, and PVMs are generated to replace dead cells, which do not have a long lifespan (all these cells have to be replaced within weeks). New blood vessels are also generated during adulthood, in both brain and meninges. All components required for angiogenesis are therefore present in the CNS. PVMs appear ideally located to synthesize and release ECM molecules nec-

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essary for angiogenesis. Similar mechanisms implicating macrophages appear to initiate and regulate adult angiogenesis (Anghelina et al. 2004; Moldovan 2002 a, b). These authors suggest that macrophages are “architects of angiogenesis”, scaffolding and preparing the orderly assemblage of newly generated endothelial cells. Other authors suggest that endothelial cells replenish from circulating bone marrow-derived monocytes and dendritic cells (Fernandez et al. 2000; Havemann et al. 2003). These authors have demonstrated in vitro that both monocytes and dendritic cells can transform into endothelial cells in the presence of angiogenic growth factors. The phenotypic overlap between monocytes and endothelial cells further supports the possibility of lineage relationships between the two cell types (Schmeisser et al. 2003). Again, the HSPG perlecan may be involved, activating the growth factors that lead to angiogenesis (Jiang et al. 2004). Whatever the case is, the paradigm that postnatal angiogenesis is exclusively caused by outgrowth of endothelial cells from preformed vessels has to be modified (Havemann et al. 2003).

6.5.2.4 Role of Macrophages in CNS Plasticity We suggest that PVMs and pial macrophages behave similarly to SEL macrophages and govern CNS plasticity, i.e. orchestrate morphological and functional changes that permit astrocytes and neurons to adapt to physiological challenges, respond to any biological requirement for a new neural architecture, and respond to injury by attempting repair. PVMs produce growth factors and ECM molecules that may participate in the assembly and dynamics of basal laminae, which serve as substrate for binding and activation of growth factors and cytokines involved in the morphological modification required for proper CNS functioning. In every part of the CNS the cells facing the basal laminae and subadjacent macrophages are astrocytes (astrocytes of the glia limitans at the brain surface and at the border of every major brain structure, perivascular astrocytes, and SEL astrocytes). The distribution of basal laminae and their potential function in brain is described by Mercier and Hatton (2004). How astrocytes are implicated in CNS plasticity is reviewed by Hatton (2004) and Mercier (2004). Astrocytes are ideally located to serve as an obligatory information relay between meningeal cells (macrophages and fibroblasts) and neurons. This is facilitated by the neural architecture, neurons never directly contacting basal laminae, except in circumventricular organs where the total retraction of astrocyte endfeet facilitates the passage of neurohormones or neuromediators. It is important to consider the possibility that fibroblasts may assist macrophages in the CNS extraparenchymal compartment in both providing information captured in the brain fluids and producing ECM molecules and growth factors appropriate to the initiation of plasticity. It has been shown that meningeal cells, both macrophages and fibroblasts, produce ECM molecules for the formation of the basal lamina (Sievers et al. 1993). Fibroblasts produce numerous collagens, growth factors (for a review, see Mercier and Hatton 2004) and morphogens such as semaphorins. Meningeal cells are involved in nerve regeneration (Shearer and Fawcett 2001) and in the formation and dynamics of the glia limi-

6.6 Conclusion: Macrophages as Architects of the CNS Throughout Adulthood

tans (Struckhoff 1995). Fibroblasts are connected to each other by their processes (Mercier and Hatton 2001) via functional gap junctions (Spray et al. 1991) to form a network throughout the CNS extraparenchyma (meninges, meningeal separations between the brain major structures, perivasculature). Gap junctional inter-fibroblast communication likely facilitates the propagation of information available from the CSF and blood to inform the neighboring macrophages. Interestingly, it has been shown that fibroblast-produced collagen-1 fibrils appear to participate in the mediation of growth factor by HSPG. Perlecan promotes FGF-2 delivery in collagen-1 fibrils (Yang et al. 2005).

6.6 Conclusion: Macrophages as Architects of the CNS Throughout Adulthood

The association of CNS macrophages with basal laminae throughout the CNS, together with the production by these cells of ECM molecules that interact with proliferating and differentiating growth factors and cytokines suggest that macrophages are involved in tissue maintenance, cell replacement, and morphological/functional plasticity in adulthood. It is tempting to conclude that macrophages achieve their architectural functions by producing and secreting appropriate signaling molecules, attracting signaling molecules secreted by other cells, and supervising the ECM/growth factors interactions that take place in the adjacent basal laminae. Regulation of the signaling molecules’ activity, presentation to neuronal and astrocytic receptors, and eventual intracellular internalization of the HSPG/growth factors complexes constitute one of the basic mechanisms by which the macrophages could influence CNS plasticity. We further suggest that PVMs and meningeal macrophages form a highly organized network throughout the CNS. Gap junction intercellular communication would allow the macrophage network to sense blood, CSF, and neurone-borne signals, analyze eventual biological deficit, tissue injury, or entry of pathogens into the CNS, produce signaling molecules, and ultimately elaborate the appropriate response to alleviate the challenge. The same organization and mechanisms may constantly serve neural plasticity, orchestrating the production, differentiation, migration, and orderly integration of new vascular and neural cells throughout the CNS, as well as controlling the growth and destruction of cell processes and synapses necessary for the dynamic activity of the CNS.

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7 The Microglial Component Ingo Bechmann, Angelika Rappert, Josef Priller, and Robert Nitsch

7.1 Microglia: Intrinsic Immune Sensor Cells of the CNS 7.1.1 Development

Microglial cells were first described by the Spanish neuroscientist del Rio-Hortega (1932). On the basis of the first selective stain for microglial cells, a weak silver carbonate method, del Rio-Hortega (1932) introduced microglial cells as a distinct entity belonging to the class of glial cells. Despite the longstanding debate in past decades over the origin of microglial cells, del Rio-Hortega’s original observation of “fountains of microglia” in the tela choroidea and the pia appears to hold true, in that leptomeningeal mesenchymal cells are indeed found to enter the neuropil, where they transform into microglia. In addition, it is now clear that monocytes provide a second source of microglia, while convincing evidence for a neuroectodermal origin is lacking. Microglial precursors populate the CNS parenchyma during early embryonic development. At that stage, they are referred to as fetal macrophages (Takahashi et al. 1989; Alliot et al. 1999a), which, like their counterparts in the blood, show a rounded morphology. Later, they develop short processes, which subsequently mature into a fully ramified shape characteristic of adult microglia. Their presence prior to vascularization of the neuroectoderm (for a review, see Kurz et al. 2004) indicates that they are not blood-borne, but rather derive from leptomeningeal cells which, in turn, derive from the yolk sac. At later stages, monocytes from the blood are also recruited and transform into microglia. Perinatally, microglial cells aggregate as clusters of so-called ameboid cells at specific locations in the brain, particularly in the corpus callosum (for a review, see Ling and Wong 1993). These microglial progenitor cells undergo substantial proliferation, migrate into the overlying cerebral cortex along fiber tracts, and differentiate into fully ramified microglia. In the adult, early studies using an injection of colloidal carbon to prelabel circulating monocytes demonstrated their recruitBlood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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ment and subsequent transformation into microglia following brain lesion (for a review, see Kaur et al. 2001). On the basis of such findings, Kaur et al. (2001) stated that: “circulating monocytes are the major source of brain macrophages in traumatic brain lesion. With the completion of their roles as scavanger cells at the site of injury, they will become microglia in the healing process. The whole process, therefore, recapitulates the microglial ontogeny in early development.” In fact, this concept has been confirmed in modern models of bone marrow transplantation (Priller et al. 2001; Bechmann et al. 2005; see Chapter 6). In the adult brain, microglia make up about 5–20% of the entire central nervous system glial cell population; and one can thus roughly calculate that there are about as many microglial cells as neurons in the adult brain.

7.1.2 Microglial Activation

In the absence of pathology, microglial cells exhibit a ramified morphology with a small cell body and long, thin processes which define their territory (Fig. 7.1). In this state, they are known as “resting” microglia. Virtually any kind of brain pathology is accompanied by a morphologic transformation of these ramified cells into what is referred to as their “activated” form. Such activated cells show stubby processes, losing the small extensions typical of the “resting” form. Once activated, they are not easy to distinguish from infiltrating mononuclear cells/ macrophages, which, in turn, develop microglia-like ramifications once inside the brain (Priller et al. 2001; Bechmann et al. 2005) under the influence of astrocytic signals (Sievers et al. 1994; Hailer et al. 1998; for a review, see Bechmann and Nitsch 2004). The induction of microglial activation as indicated by their morphologic transformation has led to the helpful concept of microglia as “sensors” of pathologic events within the CNS (Kreutzberg 1996). While there are certainly uniform phenotypic changes during microglial activation from ramified, to ameboid, and finally phagocytic, a closer look reveals remarkable changes in surface antigen expression and cytokine release in different pathologies, which certainly reflect the different activating signals. For instance, the MHC-II complex is not induced in the course of retrograde (Liu et al. 2005) axonal degeneration, but is enhanced for up to several years after anterograde (Wallerian) axonal degeneration (Kosel et al. 1997). Interestingly, such MHC-II+ microglia in zones of degeneration are highly ramified, challenging the concept that ramification always reflects a “resting” state (Bechmann et al. 2001 c). In fact, in vitro, the cytokine GM-CSF drives ramification and differentiation of a microglial subpopulation towards a dendritic cell-like state, while other cells of the same culture do not differentiate in that direction (Fischer and Reichmann 2001; Santambrogio et al. 2001). One can thus assume that the functional plasticity of microglia is as heterogeneous as the catalogue of brain disorders, and that site-specific subpopulations may exist with different capacities to differentiate in a macrophage-like or dendritic cell-like direction.

7.1 Microglia: Intrinsic Immune Sensor Cells of the CNS

Fig. 7.1 (A) Microglial cells in the normal brain. Note the typical ramified morphology of what are regarded as “resting” microglia (open arrow). Juxtavascular microglia are found to engulf small vessels (arrowheads). (B, C) (double fluorescence) Supplementation of perivascular macrophages and microglia by bone marrow cells. The marrow was transduced to express green fluorescent

protein (GFP), allowing clear-cut detection ofblood-derived cells within the brain (Priller et al. 2001; C). These cells express the microglia/ macrophage marker MAC-1 (B). A perivascular cell (white arrow) and a juxtavascular microglial cell (white arrowhead) are GFP-positive, while none of the highly ramified intraparenchymal microglia (black arrow, with white outline) appear to derive from blood.

7.1.3 Antigen Presentation/Cytotoxicity

In most circumstances, microglia are immature antigen-presenting cells (APCs; Carson et al. 1998). Only after a multi-step activation process involving the CD40-CD40 ligand, CD80-CD28 binding, as well as cytokine stimulation with GM-CSF and IFN-gamma (Matyszak et al. 1999), are they rendered competent APCs. Unless such strong activation signals are present, antigen presentation by microglia provides insufficient co-stimulation, thus inducing T cell anergy, an important mechanism of immune tolerance (for a review, see Kamradt and Mitchison 2001). The relative resistance of microglia to transformation into acti-

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vated APCs and the resulting tolerogenic antigen presentation reflect the need to be “more tolerant” within the brain, compared with less vulnerable organs, in order to minimize secondary damage, an unavoidable side-effect of any immune response (for a review, see Kwidzinski et al. 2003). In fact, besides providing help by phagocytosing degenerated material (such as myelin with its growth-inhibiting epitopes) and secreting neuroprotective molecules, microglia can also become cytotoxic, e.g. through the release of NO, IL1-beta, and TNF-alpha (Stoll et al. 2002). This is particularly prominent upon binding of LPS to Toll-like receptor 4, leading to microglia-mediated death of neurons and oligodendrocytes (Lehnardt et al. 2002, 2003). This and similar activating pathways of innate immunity may in part underlie the devastating effects of bacterial meningitis, rendering not only the infectious agent, but also the host’s microglial response against it, potential targets of therapeutic intervention.

7.2 Terminology: Subtypes and Their Location in Regard to Brain Vessels 7.2.1 Perivascular Macrophages

Since microglia share most markers with cells of the monocyte/macrophage lineage, immunocytochemical detection in situ identifies them together with extraparenchymal macrophages of the leptomeninges and the perivascular (Virchow-Robin) spaces. This latter population is sometimes referred to as perivascular microglia, although they are located outside the neuropil proper. As shown in Fig. 7.2, perivascular macrophages are also distinct from pericytes, which regularly engulf capillary endothelial cells (see Chapter 6). Perivascular macrophages accumulate degenerated myelin (Kosel et al. 1997) and seem to be particularly important for antigen presentation. In fact, during brain inflammation, T cells first reside within the perivascular spaces, where they are believed to be re-stimulated by these macrophages (Platten and Steinman 2005; see Fig. 7.3).

7.2.2 Juxtavascular and Other Microglia

The first layer of the neuropil proper is formed mainly by astrocytic endfeet building the glia limitans perivascularis, which is separated from the outer vessel wall or, where existent, the Virchow-Robin spaces by a basement membrane (for details of the perivascular space, see Fig. 7.2). Some of the microglial cells participate in forming the glia limitans (Lassmann et al. 1991) or engulf the smaller vessels of the brain (Fig. 7.1). These cells are known as juxtavascular microglia. Other microglial cells seem to have no direct contact with the vasculature and are simply referred to as microglia. In routine light microscopic sections used for neuropathol-

7.2 Terminology: Subtypes and Their Location in Regard to Brain Vessels

Fig. 7.2 (A) Location of perivascular cells (PC) in the perivascular (Virchow-Robin) spaces (PS). The dots mark the basement membranes. A first basement membrane (1) surrounds the endothelial cell (E), which in turn is surrounded by a pericyte (PY) and the second basement membrane (2). The third basement membrane (3) is located on the top of the glia limitans, which is visualized by GFAP-immunocytochemistry (arrows). The perivascular cells are located between the second and the third basement membrane and are often found to wrap small processes (open arrows) around brain vessels. Scale bar: 2 lm. (B) Perivascular space after intraventricular injection of Mini Ruby (MR). Within minutes upon injection, the tracer can be found in

the perivascular spaces (open arrows), demonstrating their connection to the subarachnoid space, where the CSF drains from the ventricles. Phagocytosis of the tracer by the perivascular macrophages (PC) is evident. Scale bar: 1 lm. (C) Mini Ruby (MR) clusters confined to the perivascular space. Following injection of Mini Ruby, the tracer remains confined to the space between the second (2) and the third basement membrane (3). Mini Ruby clusters are attached to the processes of perivascular cells (open arrows), but cannot be found within pericytes or astrocytic processes of the glia limitans. Scale bar: 0.5 lm. (Reprinted from Bechmann et al. 2001 b, copyright, with permission from Elsevier).

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Fig. 7.3 Schematic drawing of prominent cellular interactions leading from brain pathology to innate and adaptive immune responses, which may have both beneficial and detrimental effects on the neurologic outcome (for a review, see Bechmann and Nitsch 2001). Virtually any kind of brain pathology induces microglial activation, which in turn activates astrocytes. Astrocytic signaling at the glia limitans is likely to initiate adhesion molecule expression on endothelial cells. Activated microglia may also directly impact on endothelial cells, e.g. at the level of tight junctions. The VirchowRobin space represents the interface between blood and neuropil.

Upon diapedesis, leukocytes may either remain in the perivascular compartment or progress into the neuropil. Data indicate that progression across the glia limitans requires antigen presentation by perivascular macrophages (Tran et al. 2000). There is some confusion as to the nomenclature of these cells (Platten and Steinman 2005). While it seems clear now that invading monocytes transform into perivascular macrophages and further into (juxtavascular) microglia, a possible route back from the neuropil to the perivascular space and blood stream remains to be shown. tjs = tight junction, pv = perivascular.

7.3 Turnover of Brain Mononuclear Cells by Precursor Recruitment Across the BBB

ogy, pericytes, perivascular macrophages, and juxtavascular microglia cannot easily be distinguished. This is a drawback, since they certainly behave differentially and exert different functions during brain pathology. However, at the ultrastructural level, clear-cut distinction is possible (see Chapter 6) and there are antibodies to selectively label the cells (Alliot et al. 1999 b; for a review, see Guillemin and Brew 2004).

7.3 Turnover of Brain Mononuclear Cells by Precursor Recruitment Across the BBB 7.3.1 Perivascular Cells

After the early engraftment of fetal macrophages in the non-vascularized neuroectoderm, a second wave of macrophage infiltration into the brain occurs at the time of axonal pruning. In the adult, only perivascular, leptomeningeal, and choroid plexus macrophages show perpetual turnover. Perivascular macrophages are rapidly replenished by bone marrow-derived precursors with a half-life of less than 2 months in mice (Bechmann et al. 2001 a). Hickey and Kimura (1988) found substantial turnover of perivascular macrophages in lethally irradiated rats after bone marrow transplantation. The de novo invasion of macrophages into rat brain perivascular spaces occurs independent of irradiation/bone marrow transplantation, since fluorescently labeled peripheral blood monocytes find their way into brain perivascular spaces within 24 h after tail vein injection (Bechmann et al. 2001 b). Moreover, in human female sex-mismatched bone marrow transplant recipients, male donor-derived macrophages could be detected in the perivascular spaces of the brain (Unger et al. 1993). As pointed out above, there is evidence to suggest that the perivascular cells located at the interface between blood and brain participate in antigen presentation through MHC class II molecules (Hickey and Kimura 1988; Graeber and Streit 1990). Another population of immunologically active cells in the brain perivascular spaces are CD11c+ dendritic cells (DCs), which may derive from systemic circulation (Serafini et al. 2000) or from CNS-resident precursors (Santambrogio et al. 2001). A small number of MHC class II-expressing donor-derived DCs can be found in the meninges and perivascular spaces of chimeric mice after bone marrow transplantation (Greter et al. 2005). It is at present not clear whether such DCs, macrophages, or microglia can leave the brain to present antigens in lymphoid organs. The presence of brain antigens in cervical lymph nodes during autoimmune neuroinflammation (de Vos et al. 2002) may thus be due to antigen drainage within the cerebral spinal fluid (CSF) or carriage by migratory cells.

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7.3.2 Microglia

In contrast to the high turnover of perivascular and meningeal macrophages, there is general consensus that parenchymal microglia are slowly replenished by bone marrow-derived precursors in the adult. In fact, several studies found negligible engraftment of microglia in rat or mouse brains after lethal irradiation and bone marrow transplantation (Hickey and Kimura 1988; Lassmann et al. 1993; Greter et al. 2005). Using novel molecular biological tools, such as the genetic modification of hematopoietic stem cells with a green fluorescent protein, Priller et al. (2001) described slow, but significant microglial engraftment in mice. These findings were later confirmed by several independent studies (Vallieres and Sawchenko 2003; Hess et al. 2004; Simard and Rivest 2004). In some regions of the brain, microglial engraftment was particularly high; and up to a quarter of the entire microglial population was donor-derived within 4 months (Priller et al. 2001). Interestingly, bone marrow-derived microglia appear to be specifically recruited to sites of CNS pathology. Thus, focal cerebral ischemia and facial nerve axotomy significantly enhanced microglial engraftment in rat and mouse brain (Flugel et al. 2001; Priller et al. 2001). Moreover, circulating monocytic cells from spleen were found to infiltrate layers of axonal degeneration after entorhinal cortex lesion, where they rapidly acquired a microglial morphology (Bechmann et al. 2005). These findings demonstrate that microglial engraftment can occur, independent of irradiation and bone marrow transplantation.

7.3.3 Turnover Used by “Trojan Horses”

An important clinical example of the relevance of macrophage turnover in the brain is the neuropathogenesis of AIDS (for a review, see Gonzalez-Scarano and Martin-Garcia 2005). According to the “Trojan horse” hypothesis, the lentivirus enters the CNS in myeloid cells trafficking to the brain after systemic infection. Perivascular macrophages and to a lesser extent microglia are the most important cell types infected by HIV-1 in the CNS. Therefore, it is not surprising that pathological changes in the brain correlate with the presence of activated bone marrow-derived cells. A number of chemokines and their receptors are induced in HIV encephalitis; and these control the recruitment of leukocytes into the brain (Schmidtmayerova et al. 1996; for a review, see Ransohoff et al. 2003). Therefore, the mechanisms of leukocyte passage across the BBB will be reviewed below.

7.4 Microglial Impact on BBB Function

7.4 Microglial Impact on BBB Function 7.4.1 Concept of the BBB

Paul Ehrlich’s discovery (1885) that systemically injected water-soluble dyes do not reach the neuropil led to the concept of the “Bluthirnschranke” (Lewandowsky 1900), providing a mechanical barrier from blood to brain. Endothelial tight junctions were later identified as the structural correlate of this function. This concept has also been used to explain the early notion that leukocytes are rare within the brain and, thus, that the architecture of brain microvessels’ endothelium maintains its immune-privileged status (for reviews, see HarlingBerg et al. 1999; Pachter et al. 2003; Bechmann 2005). It is noteworthy that the barrier function has been described at the level of brain capillaries (Reese and Karnovsky 1967; for a review, see Ge et al. 2005), while leukocyte diapedesis takes place in postcapillary venules. The original observation underlying the term BBB thus applies to sites and features of the vascular arbor other than those involved in leukocyte recruitment. In fact, leakage of the BBB need not necessarily come along with inflammatory infiltrates (see Chapter 24). It has also been shown that inflammatory reactions in the CNS can lead to the influx of inflammatory cells, probably before BBB breakdown, and areas of inflammation do not correlate with those of greater permeability (Muller et al. 2005). A correlation between monocyte infiltration and loss of the tight junction protein ZO-1 has been reported (Boven et al. 2000), but there is also recent evidence for leukocytes traversing microvessels via a transcellular rather than a paracellular route, thus leaving tight junctions intact (Wolburg et al. 2004). However, activated T lymphocytes in search of their specific antigens can freely cross the endothelial wall in the absence of pathology. This appears to happen in a random fashion, a process known as immunological surveillance (Hickey 2001). During the past decade, trafficking signals which guide the transmigration of leukocytes into the brain as well as their migratory routes have been identified (for a review, see Randsohoff et al. 2003). Chemokines are likely to be such signaling molecules because of their specificity in mononuclear leukocyte migration (Brown 2001). The microglial cell constitutes a potent source of these molecules; and its role as a sensor of pathologic changes within the neuropil makes it tempting to speculate that microglia initiate and regulate the recruitment of specific leukocyte populations to areas of degeneration by inducing the expression of chemoattractant and adhesion molecules on endothelial cells. In the following, we thus provide a brief overview of chemokine families, as well as functional data identifying some candidates for microglia-mediated changes at the level of the brain endothelium.

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7.4.2 Chemokines – an Overview

Chemokines are a diverse family (43 human chemokines are known today) of chemotaxis-inducing cytokines. They are small peptides (8–10 kDa) and have been found to be involved in the pathogenesis of many neuroinflammatory diseases, ranging from multiple sclerosis (MS) and stroke to HIV encephalopathy. The chemokine family is currently divided into four groups, depending on the number and spacing of the conserved cysteines in the protein sequence (Baggiolini et al. 1997; Luster 1998). The C-X-C family (also called alpha) has a single amino acid between the initial two cysteines. If the first two cysteines are adjacent to each other, the peptides are classified in the C-C family (also called beta), while the unique gamma chemokine or C chemokine (lymphotactin) has lost two of the four conserved cysteines (Cys-2, Cys-4). Finally, in the CX3C family, the chemokines have three amino acid residues separating the first two cysteines in the sequence. The CX3C chemokines are the only membrane-bound chemokines. Chemokines mediate their biological activities through 19 different G protein-coupled cell surface receptors. The chemokine receptors are named according to their chemokine subfamily classification. Chemokine receptors are promiscuous in that they can bind to more than one chemokine, and more than one chemokine can often bind to the same receptor.

7.4.3 GAG/Duffy

The presence of chemokine-binding sites on human brain microvessels (Andjelkovic et al. 1999) suggests that chemokines produced locally by microglia, astrocytes, and perivascular cells will either diffuse or be transported to the endothelial cell surface, where they will be immobilized for presentation to leukocytes. However, in vivo chemokines interact with sulfate sugars of the glycosaminoglycan (GAG) family, such as heparan sulfate and chondroitin sulfate, which can limit their dissemination. GAGs decorate proteins in the extracellular matrix and on the cell surface and not only interfere with chemokine diffusion, but also provide highly specific substrates for the presentation of chemokines to other cells, e.g. on the luminal surface of endothelial cells. Hence, they are appropriately positioned to activate leukocytes in close proximity (Middleton et al. 2002; Rot and von Andrian 2004). Tissue-derived chemokines can cross the endothelial cell barrier passively through intercellular junctions (Song and Pachter 2004) or can be transported (Middelton et al. 1997; Rot and von Andrian 2004). One molecule is expressed by endothelial cells and may contribute to chemokine transport: the Duffy antigen (also known as the Duffy antigen receptor for chemokines, or DARC), which binds numerous chemokines (Nibbs et al. 2003).


7.4.4 Chemokine Expression in the CNS

Various chemokine receptors of the different chemokine families are expressed by different types of brain cells, neurons, astrocytes, and microglia. However, with the exception of CX3CL1 (fractalkine), all chemokines found in brain tissue are expressed by glial cells and infiltrating leukocytes. Both astrocytes and juxtavascular microglia are in physical proximity to the endothelium. Pericytes and perivascular macrophages are widely distributed at the level of the BBB. Thus, these cells are in a prime position to influence the transmigration of leukocytes. In addition, they are established sources of soluble molecules that can enhance vascular permeability. Most information concerning the migration of lymphocytes into the neuropil has emerged from studies using experimental allergic encephalomyelitis (EAE), the animal model of multiple sclerosis (MS), and HIV infection. In a state of health, very few leukocytes infiltrate the brain (Ludowyk et al. 1992); but in disorders such as MS the “barrier” becomes permissive, resulting in intense infiltration of the CNS by T lymphocytes, whose subsequent activity appears to underlie the onset and progression of disease (Brown 2001). In patients with HIV encephalitides and MS or in EAE tissue of animals, several chemokines, such as CCL2 (monocyte chemoattractant protein, MCP-1), CCL3 (macrophage inflammatory protein 1a, MIP-1a), CCL5 (RANTES), and CXCL10 (interferon-inducible protein 10), can be detected in the CSF (Letendre et al. 1999; Sorensen et al. 1999). The corresponding receptors CCR5 and CXCR3 are expressed by activated T cells, which are increased in the peripheral blood, CSF, and lesions of MS patients (Misu et al. 2001). These findings lead to the idea that these receptors and their ligands are important for lymphocyte trafficking into the CNS during disease. In fact, all these chemokines are expressed by microglia (Kremlev et al. 2004; Rock et al. 2004).

7.4.5 CCL2 and CCR2

CCL2 and its receptor CCR2 have been implicated as key mediators of leukocyte entry into the CNS, since CCL2 expression has been found in virtually all forms of CNS insult, including acquired immunodeficiency syndrome (AIDS) with dementia (Cinque et al. 1998; Mengozzi et al. 1999; Weiss et al. 1999) and EAE (Ransohoff et al. 1993; Mahad and Ransohoff 2003). CCL2 is produced by macrophages, microglia, activated astrocytes, perivascular macrophages, and endothelial cells (Sozzani et al. 1995; Rollins 1996; Simpson et al. 1998; Hofmann et al. 2002; Babcock et al. 2003). It potently recruits monocytes and T cells into the brain (Sozzani et al. 1995; Rollins 1996). By using a coculture model of the BBB, Weiss and co-workers demonstrated that 90% monocytes and 10% lymphocytes transmigrate in response to CCL2, while the input population of mononuclear cells consisted of 90% lymphocytes and 10% monocytes (Weiss et

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al. 1998). Neutralizing antibodies specific for CCL2 ameliorated the progression of EAE disease (Karpus et al. 1997). Mice lacking either the corresponding receptor CCR2 or one of its ligands were relatively resistant to the development of EAE, despite having T cell responses equivalent to those observed in wild-type mice (Fife et al. 2000; Izikson et al. 2000; Huang et al. 2001). CNS tissues of CCL2-deficient mice were virtually devoid of monocytes at time-points equivalent to those at which wild-type mice developed neurological impairment (Huang et al. 2001). CCR2 was also essential for the accumulation of monocytes at the epicenter of a spinal cord contusion; and their absence in CCR2-deficient mice was associated with impaired clearance of tissue debris (Ma et al. 2002). In zones of axonal degeneration, CCR2 deficiency also completely blocked the invasion of leukocytes (Babcock et al. 2003), which normally takes place within the first days after injury (Bechmann et al. 2001 c, 2005). Nevertheless, controversial data are found in humans. The CCL2 concentration in the CSF of patients with early active MS or during relapses is reduced (Sorensen et al. 2004). However, these data did not exclude a role of CCL2 in other phases of MS. Thus, further studies directly in MS brain tissue could clarify the role of CCL2 in MS.

7.4.6 CCL3 and CCL5

Of the CSF chemokines, only CCL3 and CCL5 are associated with enhanced migration of T lymphocytes from MS patients, an effect that is ascribed to the over-expression of CCR5 on these cells (Zang et al. 2000). CCL3 is expressed by perivascular macrophages (Hofmann et al. 2002). Neutralizing antibodies and targeted gene deletion in animal models of CNS inflammation have yielded conflicting results about the roles of these chemokines. Inactivation of CCR5 in acute viral encephalitis was effective in decreasing the numbers of infiltrating leukocytes (Glass et al. 2001). In contrast, loss of CCR5 activity in mouse models of MS was without effect; and in MS it may slow disease progression (Tran et al. 2000; Kantor et al. 2003). While neutralizing antibodies specific for CCL3 ameliorated the progression of EAE (Karpus et al. 1995), CCL3 knockout mice were entirely susceptible to EAE (Tran et al. 2000). Thus, these findings leave the specific functions of these chemokines in these disease models unresolved.

7.4.7 CXCR3 and CXCL10

Numerous studies utilizing animal models and transgenic approaches have supported a role for CXCL10 and its receptor CXCR3 in the trafficking of lymphocytes during acute CNS inflammation. Under inflammatory conditions, both CXCL10- and CXCR3-expressing T cells could be found in the CSF (Sorensen et


al. 1999). CXCR3 expression is not specific to T cells in the brain, but can also be found on other populations outside the neuropil. However, if the ligand CXCL10 appears in the CSF, CXCR3-expressing T cells stop recirculating in the periphery (Trebst and Ransohoff 2001). In addition, CXCL10-CXCR3 is probably also involved in the transmigration into the CNS parenchyma: Sorensen could demonstrate a direct correlation between CXCL10 expression and the number of CXCR3-expressing cells (Sorensen et al. 2002). CXCL10 is produced by astrocytes, perivascular macrophages (Karpus and Ransohoff 1998), and microglia (Lokensgard et al. 2001), and may establish the gradient necessary for the transmigration of T cells into the CNS parenchyma. Neutralizing antibodies specific for CXCL10 may ameliorate the progression of disease by inhibiting mononuclear infiltration (Fife et al. 2001). In contrast, Narumi et al. (2002) reported an exacerbation of EAE progression by applying similarly utilized CXCL10-neutralizing antibodies. Their finding is supported by studies using CXCL10 knockout mice, which were entirely susceptible to EAE (Klein et al. 2004). Interestingly, CXCL10 expression in the CNS leads to an increase in leukcocyte infiltration; however, these infiltrates are restricted to the meninges and perivascular compartments (Boztug et al. 2002; Trifilo and Lane 2003). This indicates that CXCL10 could be a signal which directs lymphocytes across the endothelial layer, but not beyond, i.e. across the glia limitans (see Fig. 7.2).

7.4.8 Microglia-Endothelial Cell Dialogue

All these results support the view that chemokines are crucial in lymphocyte trafficking during established CNS autoimmunity. Their individual role in attracting particular subsets of leukocytes such as T and B lymphocytes, monocytes, or NK cells, as well as the signals initiating chemokine expression during pathology have yet to be identified. Some controversy in the results probably reflects that chemokines and their receptors are promiscuous, given that by blocking one chemokine, others compensate for the effect (Klein et al. 2004). However, by using conditional and cell type-specific knockout strategies, a picture may eventually evolve of how microglia and perivascular macrophages attract particular subtypes of leukocytes into particular regions of the CNS (Rock et al. 2004). It now seems clear that the dialogue between microglia and brain endothelial cells plays a crucial role in the process.

7.4.9 Microglial Effects on Tight Junctions

Inflammation not only involves the recruitment of leukocytes, but also the opening of endothelial barriers in order to allow serum molecules such as antibodies and complement factors to gain access to areas of degeneration. Activated micro-

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glia produce cytokines and other toxic factors promoting neurodegeneration and activation of other glial cells, including astrocytes. It is likely that microgliamediated astrocytic activation also affects BBB integrity through crosstalk with astrocytes and perivascular cells. In post mortem studies of HIV-1-associated dementia, the presence of damaged tight junctions, indicated by fragmentation or absence of immunoreactivity for occludin and ZO-1, correlated with levels of monocyte infiltration (Dallasta et al. 1999; Boven et al. 2000). The coincidence of the greatest number of abnormal junctions with the highest levels of microglial activation in a study of active MS suggests that some of the tight junction abnormalities result from the pathophysiological action of cytokines, matrix metalloproteinases, and other immune effectors present in the disordered milieu of the MS lesion (Kieseier et al. 1999; Plumb et al. 2002). This apparently also applies to perivascular macrophages: in experimental simian immunodeficiency virus encephalitis, the accumulation of these cells correlates with the fragmentation and decreased immunoreactivity of ZO-1 and occludin (Luabeya et al. 2000). Several lines of evidence indicate that locally produced microglia-derived cytokines, in particular TNF-a, are the most likely candidates for mediating tight junction disruption (Gloor et al. 2001; Poritz et al. 2004; Wang et al. 2005). TNF-a and interferon-c induce a striking fragmentation of ZO-1 via F-actin rearrangement in cultures of microvascular endothelial cells (Blum et al. 1997). Indeed, high levels of these cytokines are present in the CNS of patients with HIV-1-associated dementia complex (Griffin 1997), and microglia are a potent source of TNF-a (Benveniste 1997; Persidsky et al. 2000). These findings could explain how microglial cells control the opening and closing of tight junctions, thus fostering permeability for molecules from the blood and possibly the infiltration of leukocytes.

7.5 Concluding Remarks

Sound data indicate that microglia, once activated by pathologic changes in their surrounding milieu, secrete chemokines and cytokines which impact endothelial cells at two levels: the expression of adhesion/chemoattractant molecules and the maintenance of tight junctions. While the former clearly is related to cell type-specific leukocyte recruitment, it is unclear as to how far tight junction changes are involved in initiating and facilitating the infiltration of leukocytes. In vitro data are difficult to interpret, since endothelial cells derive from various sites of the vascular arbor and there is a lack of sound studies specifically addressing changes in tight junctions at the level of postcapillary venules, where diapedesis takes place. Recently, trans-rather than paracellular diapedesis has been shown, using serial electron microscopic sections from venules of EAE brains (Wolburg et al. 2004), suggesting that diapedesis is independent of the maintenance of tight junctions. It is, however, clear that the permeability of the BBB is enhanced during neuroinflammation; and this may well allow serum molecules involved in the inflammatory cascade to gain access.

References

Microglia as early sensors of pathological events within the neuropil (Kreutzberg 1996) are likely to initiate innate and adaptive immune responses by cytokine and chemokine signaling. Be it directly or via astrocytes forming the glia limitans, this will induce endothelial expression of adhesion molecules, allowing leukocyte recruitment. There are also indications that microglia institute tight junctional changes, thereby fostering at least BBB permeability. Beyond this, liberation of cells from their tight junctions may provide a more general mechanism of inflammatory cascades by supporting migration into sites of injury. The current challenge is to identify the crucial signaling molecules from microglia to endothelial cells, which may provide promising targets for interfering with undesired (autoimmune) neuroinflammation.

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8 The Bipolar Astrocyte: Polarized Features of Astrocytic Glia Underlying Physiology, with Particular Reference to the Blood-Brain Barrier N. Joan Abbott

8.1 Introduction

During the development of the mammalian central nervous system (CNS) from the simple neural tube, astrocytes derived from multipotent precursor cells frequently show a transition from a bipolar to a multipolar form, i.e. from a columnar or spindle-shaped cell with a single process at either end of the cell body to a cell with multiple processes, including the highly branched phenotype that gave rise to the term “astrocyte” (star-shaped or stellate cells). The bipolar phenotype, with distinct apical and basal poles at opposite ends of the cell derives from the epithelial organization and function of the neural tube. In the mature CNS, astrocytes with a wide variety of morphologies can be observed, but interestingly, many of them preserve aspects of a polarized phenotype, even when the apical-basal (bipolar) distinction is less clear. The mammalian bloodbrain barrier (BBB) formed by the brain endothelium is induced and maintained by chemical factors derived from the underlying neural tissue, and the polarized processes of astrocytes associated with the endothelium are responsible for the induction of a number of features of the BBB phenotype. Moreover, polarized signaling between astrocytes and endothelium is important in the modulation of BBB function. This chapter reviews the origin of polarity in the astrocyte population and ways in which this polarity is crucial to function, with particular emphasis on the BBB interface.

8.2 Formation of the Neural Tube

The CNS of vertebrates is formed from the ectoderm of the embryo. In the process of “neurulation”, the dorsal ectoderm (skin) folds inwards to create the neural plate along the longitudinal axis, followed by fusion of the lips of the plate (neural folds) to form the neural tube (Fig. 8.1). The neural tube then separates Blood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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Fig. 8.1 Apical-basal polarity set up during neurulation. During development, the mammalian CNS forms by invagination of the surface ectoderm of the neural plate (A), which subsequently seals up and sinks below the surface to form the neural tube (B). The lips of the neural plate (neural folds) become the migratory population of neural crest cells, which contribute to the peripheral nervous system.

and sinks below the skin, with cells from the edges of the fold (neural crest) forming a migratory population and giving rise to many cells of the peripheral nervous system [1]. The cells of the skin ectoderm, like all epithelial layers forming an interface between tissue and a fluid-filled space (here the external environment) express a strong polarity, the apical cell membrane facing the fluid space, and the basal cell membrane secreting and apposed to a layer of specialized extracellular matrix, the basal lamina/basement membrane. This apical-basal polarity is retained in the neuroepithelial cells forming the singlelayered neural tube (Fig. 8.1). As with many epithelia facing an internalized fluid cavity (e.g. small intestine, kidney tubule), the apical membrane surface area may be increased by the presence of fine processes (cilia, microvilli).

8.3 Origin of Neurons and Glia

The neuroepithelial cells are capable of forming both neurons and macroglia – the latter comprising oligodendrocytes, astrocytes, and ependymoglia [2]. Cell division among the earliest neuroepithelial cells forms the ventricular zone (VZ), a pseudostratified epithelium adjacent to the lumen of the neural tube [3]. Within the early VZ can be observed a prominent population of radially orientated cells spanning the thickness of the neural tube, which show many characteristics in common with mature astrocytes, including the expression of GLAST-type glutamate transporters, brain lipid-binding protein (BLBP), and the glial-fibrillary acidic protein GFAP [4]. This class of cells has been called the radial glia (RG); and they help

8.3 Origin of Neurons and Glia

guide the migration of immature neurons to the cortex. The RG successively replace the neuroepithelial cells and represent more fate-restricted progenitors, most RG giving risc to a single cell type, either astrocytes, oligodentrocytes or neurons [4]. Meanwhile, cells dividing within the VZ migrate into the adjacent subventricular zone (SVZ), which in turn becomes the major germinal site. Early neuroblasts in the SVZ first generate a population of neurons, before activation of a molecular “germinal switch” leads to the later production of oligodendrocytes and astrocytes [5, 6]. Recent evidence suggests that within the spinal cord astrocytes and oligodendrocytes are derived from different precursor populations, with oligodendrocytes arising from oligodendrocyte precursor cells (OLPs) in the ventral pMN domain, while astrocytes arise from more dorsal (p2) domains [6, 7]. Thus, the generation of astrocytes occurs in two distinct phases during embryonic and fetal stages of development, deriving initially from the radial glia, and later from migratory progenitors that emerge from the dorsolateral SVZ to colonize adjacent gray and white matter. The precise sequence of events and mechanisms responsible for the generation of neurons, oligodendrocytes, and astrocytes is not yet fully understood, but it is already known to depend on the location within the CNS [8]. The germinal switch from neurons to glia is determined by a combination of intrinsic (genetic) and extrinsic signals, with some reciprocal interactions, e.g. forced expression of the oligodendrocyte gene Olig2 inhibits astrocyte differentiation, and forced expression of the activated Notch receptor Notch1a promotes formation of the astrocyte lineage, including RG and Müller cells in the retina [6, 9]. The sequence of events involved in the neuron-glial switch is better understood for oligodendrocytes than astrocytes, as more marker proteins for the stages of development and differentiation have been identified for oligodendrocytes. The major phase of neurogenesis (birth of neurons) occurs before birth in most mammals, with neuronal precursors migrating along the scaffold formed by RG to their final positions, where synapse formation and connectivity are established. In the adult, the sites of neurogenesis from endogenous precursors are predominantly confined to two major sites – the SVZ giving rise to the rostral migratory stream (RMS) of neurons migrating to the olfactory bulbs, and the hilus of the dentate gyrus generating cells for adjacent regions of the hippocampus [3, 10]. The early-appearing radial glia have a clear bipolar morphology, with the basal membrane forming the glia limitans adjacent to the pia at the outer surface of the brain and the apical membrane facing the fluid-filled cavity of the neural tube that will become the adult ventricular system [2]. Once the major wave of neuronal migration is complete, the majority of RG retract one or both processes from the inner and outer brain surfaces, develop a more branching morphology, and begin to express other features of differentiated astrocytes [11], while processes from the basolateral parts of the cell can contact a number of cell types, from adjacent astrocytes to capillary endothelial cells to neurons. An idealized ependymoglial cell showing these contacts is illustrated in Fig. 8.2 [12]. Interestingly, it was recently found that the RG guiding radial neuronal migration later extend tangentially running basal processes that channel the mi-

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Fig. 8.2 Idealized ependymoglial cell: diagrammatic representation of apical and basal processes from a “generic” ependymoglial cell derived from radial glia, showing the potential for cell-cell interactions. The apical process (I) initially makes contact with the ventricle, but may later retract. Basal or baso-lateral processes may contact a number of other structures and cell types, including the basal lamina at ectoderm-mesoderm interfaces at the pial surface (IIa) and blood vessels (IIb), and neuronal somata (IIIa), synapses (IIIb), or axons, especially in the region of the axon hillock and nodes of Ranvier (IIIc). (Modified from [12] with permission).

gration of the RMS [13]. Even more intriguingly, the migrating neuroblasts that form a moving chain of cells destined for the olfactory bulb express a number of glial features [14]. Thus, the glial cells of these germinal zones may provide both the scaffold and the migratory population – suggesting that these cells retain a multipotent phenotype. The cell layer left lining the ventricular cavity after formation of the SVZ is termed the ependyma, and the third class of macroglia, the ependymoglia, express some glial features and retain contact with this CSF-containing space. They include the tanycytes of the mature CNS and the Müller cells of the retina [2].

8.4 Morphology of Glial Polarity in Adult CNS

8.4 Morphology of Glial Polarity in Adult CNS

Within the adult central nervous system, a variety of different astrocyte morphologies can be observed (Fig. 8.3). Reichenbach [2] illustrated 11 different astrocytic-ependymoglial types, based on location and morphology; and, interestingly, at least eight of these make specialized contacts on cerebral microvessels. As immunocytochemical characterization of these glia becomes more complete, it will be possible to establish whether the differences in morphology and contacts relate to differences in expression of marker proteins, and more importantly, differences in function. In a number of locations in the adult CNS, cells related to RG or ependyma retain a clear bipolar morphology. Thus, ependymal cells themselves generally

Fig. 8.3 Semi-schematic survey of the main types of astroglial and ependymoglial cells and their localization in different layers/specialized regions of the central nervous system. (I) Tanycyte (a, pial; b, vascular), (II) radial astrocyte (Bergmann glial cell), (III) marginal astrocyte, (IV) protoplasmic astrocyte, (V) velate astrocyte, (VI) fibrous astrocyte, (VII) perivascular astrocyte, (VIII) interlaminar astrocyte, (IX) immature astrocyte/glioblast, (X) ependymocyte, (XI) choroid plexus cell. (From [2] with permission).

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have extensive apical microvilli contacting the ventricular CSF and a basal membrane associated with a prominent basal lamina, also called the “subependymal basement membrane” or basal membrane labyrinth (Fig. 8.3) [15, 16]. The specialized ependymal cells forming the choroid plexus (CP) epithelium show similar morphology and polarity, but the space between the basal laminae of epithelium and capillaries is filled with a “stromal” extracellular matrix. In the eye, both the retinal receptor layer and the retinal pigment epithelium (RPE) are ependymal derivatives, with their apical surfaces apposed to each other across the restricted remnant of the CSF-containing space between them [17]. Tanycytes are glia that retain an apical connection to the CSF, while the basal process makes contact either with other tanycytes or with capillaries. They are prominent components of neurosecretory zones (e.g. lining the floor of the third ventricle) [18]. Müller cells are effectively the RG of the retina. Depending on the species, the apical microvilli of Müller cells are more or less prominent and a well developed basal end foot faces the vitreous [2]. Where the apical connection to the ventricular or other fluid space is lost, the glial apical-basal polarity is less clear-cut (Fig. 8.2) but the basal specialisations, especially end feet on blood vessels and the glia limitans (Figs. 8.3 and 8.4) show the retained polarity.

Fig. 8.4 Apical-basal polarity of ependymoglia and astrocytes in the adult CNS. In tanycytes, the apical process remains in contact with the CSF of the ventricles, while the basolateral processes may contact cells within the parenchyma, at the pial surface, and forming blood vessels. More commonly, the apical process is reduced, separating from the ependymal cell lining the ventricle, while the basolateral processes are retained.

8.5 Astrocyte Spacing and Boundary Layers

There appears to be some plasticity between the bipolar and multipolar astrocytic forms. Moreover, some agents capable of triggering the change have been identified. Thus, transforming growth factor alpha (TGFa) is able to induce the transformation of polygonal astrocytes into a bipolar (RG-like) form in culture, with associated increase in the expression of GFAP and a reduction in motility [9]. In vivo, branched forms may revert to the bipolar form under some conditions; thus, damage and grafting may cause local stellate astrocytes to assume the bipolar shape [19], implying considerable plasticity. A subset of glial progenitor cells (and tumors) express gp130, important in glial differentiation, including conversion from multipolar to bipolar morphology [20]. Elevation of Dcyclin-associated kinases (cdk) is seen in some gliomas; and transfection to elevate cdk6 (but not cdk4) results in a change from multipolar to bipolar form [21]. There is remarkable sexual dimorphism in the arcuate nucleus of the hypothalamus, with complex stellate astrocytes in males and simpler bipolar cells in females – likely resulting from differences in gonadal steroid exposure during development and related to sexually dimorphic regulation of neuroendocrine secretions from the pituitary in the adult [22]. A population of bipolar astrocytes (GFAP-ve, vimentin +ve) is present in actively remyelinating MS lesions [23]. Taken together, these studies indicate considerable plasticity in morphology, even within the adult astrocyte population, and reversion to a less differentiated bipolar phenotype under some pathological conditions.

8.5 Astrocyte Spacing and Boundary Layers

Even in the earliest camera lucida drawings of brain sections by Lenhossek [24] and Golgi [25] (see [26]), the astrocytes appear to show a rather regular spacing, but given the fact that the Golgi impregnation method of silver staining fills only a proportion of cells present, the significance of this was unclear. In culture, it is observed that astrocytes go through a series of morphological changes, from bipolar (generally non-contacting) to stellate (contacting), including the extension and contraction of processes that result in regular spacing [27]. Astrocytes at laminar boundaries separating the association/commissural and perforant path afferents of the hippocampal dentate gyrus show a more polarized morphology than the stellate astrocytes typical of non-boundary zones, although there is no simple relation to the morphology of the laminar boundary [28]. Such studies suggest that cell morphology, polarity, and territory is influenced by interactions of astrocytes with each other and with extracellular molecules defining tissue boundaries. The result is not only morphological polarisation appropriate to function, but also an appropriate spacing that allows individual astrocytes to control a defined three-dimensional sector of parenchymal space [29, 30].

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8.6 Origin and Molecular Basis of Cell Polarity

Cell polarity is traditionally seen as a segregation of protein and lipid repertoires, i.e. distinct sets of membrane proteins (receptors, ion channels, transporters, adhesion molecules) specific for a particular cellular domain [31]. In epithelia, the clear apical/basolateral polarity is established and maintained by a number of mechanisms [32–34]: (1) cell-cell contact is sufficient to trigger the segregation of marker proteins of the apical and basolateral membrane domains to distinct regions of the membrane, (2) cell-cell contact induces association of tight junction proteins, including ZO-1, to the apex of the lateral membrane and the transmembrane proteins act as a diffusional barrier (“fence”), restricting the migration of proteins between domains, (3) establishment of the epithelial axis requires the formation of a basal lamina and cell-substratum contact, and (4) reversal of cell polarity can occur if the apical side of the cell is presented with an appropriate extracellular matrix. Rho GTPases, including cdc42, are important in establishing cell polarity; and it was recently found that the Par3/ Par6/aPKC complex acts as an evolutionarily conserved polarization signal; and a dynamic feedback loop at the apical junctional complex with Scrib/Dig/Lgl and Crumbs/Pals1/PATJ complexes organizes and defines the boundaries of polarized domains [35]. Within the adult mammalian CNS, ependymoglial derivatives make tight junction-coupled barrier layers at a number of locations, including the specialized ependyma overlying circumventricular organs (CVO), tanycytes enclosing neurosecretory zones, the choroid plexus epithelium, and the retinal pigment epithelium (Fig. 8.5). The strong apical-basal morphological polarity of these layers reflects their functional polarity (see below). In cells which do not have tight junctions, polarity can be defined by stabilization of protein complexes at the membrane by scaffolding proteins and selective targeting of proteins along the secretory or endosomal pathways, e.g. involving localization of the Golgi apparatus to the apical region for polarized secretion [31]. Thus, in neurons, significant polarization between dendritic and axonal domains can be achieved, and even without tight junctions, specialization of the membrane and its protein complexes in the region of the axon initial segment can act as a barrier to diffusion within the membrane. Further specialization can occur even within one domain, e.g. subcompartments within the dendritic tree. In migrating astrocytes, mPar6 and PKCzeta are localized to the leading edge of the cell; and they reorient the microtubule organizing center (MTOC) to the side of the nucleus facing the direction of migration [36, 37]. Cdc42 acts through the Par3/Par6/aPKCzeta complex to phosphorylate and hence inhibit glycogen synthase kinase 3b (GSK-3b), allowing association of the tumor suppressor protein adenomatous polyposis coli (APC) with microtubules, stabilizing the leading edge [37]. This generates a new polarity independent of the original polarity of cells within the neural tube.

8.7 Functional Polarity of Astrocytes and Other Ependymoglial Derivatives

Fig. 8.5 Ependymoglial derivatives coupled by tight junctions and contributing to barrier layers in the adult mammalian CNS. (1) Ependyma overlying neurosecretory zones (circumventricular organs) such as the median eminence, (2) tanycytes surrounding neurosecretory zones, (3) choroid plexus epithelial cells, (4) retinal pigment epithelium. Capillaries with tight endothelium are shown as circles with solid line, leaky endothelium with dashed line. (Based on [137], with permission).

8.7 Functional Polarity of Astrocytes and Other Ependymoglial Derivatives

Epithelial cell layers characterized by occluding tight junctions and significant transepithelial fluid movement can show functional polarization of two main types: secretory, with a net movement of fluid and solutes from basal to apical side (e.g. salivary gland acinus), and absorptive, with net movement of solutes and fluid from apical to basal side (e.g. kidney proximal tubule) [38]. A number of mechanisms are available for generating the solute and fluid movements; and individual solutes may be transported against the fluid stream. Most epithelial cells use a basolateral Na,K,ATPase to generate a Na+-K+ ionic gradient across the cell membrane. The subsequent direction and composition of the secretory or absorptive flow is regulated by the differential distribution of apical and basal ion channels and transporters, and where large volume flows occur, by the presence of aquaporin (AQP) water channels. In the choroid plexus epithelium, as for other neuroepithelial cells, the Na,K,ATPase is on the apical side and drives a Na+, Cl– and HCO–3 rich secretion of CSF into the ventricles, with water flows mediated by apical AQP1 channels [39]. Several mechanisms acting together give rise to a net absorption of K+, from CSF to blood. In the retinal pigment epithelium (RPE), with a similarly polarized transporter distribution, the movement of water must be in the opposite direction to maintain the close physical and functional association of receptors and RPE and to avoid retinal detachment [40]. The high

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density of AQP4 on the (basal) end feet of astrocytes on capillaries and at the pial surface suggests the potential for significant water flux via astrocytes, both constitutively and following neural activity, from the parenchyma to the perivascular space and the CSF of the subarachnoid space [41, 42]. The brain endothelium has undetectable AQP1 and only very low levels of AQP4 [43], consistent with this being a large surface area membrane specialized for regulating molecular flux rather than sustaining large water movements [44]. Hence, under normal conditions, it is unlikely that transendothelial flux represents a significant mode of water clearance. Instead, the perivascular basal lamina at the capillary level, connecting with perivascular (Virchow-Robin) spaces around larger vessels may be a major route for water clearance from the parenchyma, with the CSF of the subarachnoid space being the sink. However, given that the total number of AQP4 channels on fine astrocytic processes within the neuropil is about equal to those on perivascular end feet [41], a significant water flux is also likely through the parenchyma, some of which may exit across the ependyma into the ventricular CSF. Within the neuropil, extracellular water movements are expected to be mainly by diffusion, intercellular clefts being too narrow to permit significant convective (bulk) flow. However, bulk flow has been documented along the wider (low resistance) pathways created by axon tracts and perivascular spaces [44]. Within the population of processes from highly branched stellate astrocytes, it is frequently possible to observe processes on both neurons and blood vessels. A number of proteins are clustered on the perivascular end feet, including AQP4, Kir 4.1, and rSlo KCa channels [45]. Astrocytes and Müller cells often express different classes of transmitter transporters compared with neurons, and although not specifically documented, these would be expected to be mainly on the neuron-facing processes of the glia – thus, while most retinal neurons express GLT1v and EAAC1 glutamate transporters, those on astrocytes and Müller cells are predominantly GLAST [46]. Astrocytes have also been shown to express GAT1 and 2/3 GABA transporters [47]. Limitrin is a new member of the transmembrane-type immunoglobulin superfamily specifically localized to the polarized astroglial end feet and glia limitans [48]. A number of anchoring proteins are responsible for the polarized clustering of proteins in the BBB (abluminal (basal) membrane of brain endothelial cells), the blood-CSF barrier, and the perivascular end feet of astrocytes. Recent studies indicate the particular importance of utrophin in the basolateral membrane of the choroid plexus epithelium and vascular endothelial cells, while the short Cterminal isoform of dystrophin (Dp71) is localized to the glial end feet [49]. Both proteins act as anchors for the dystrophin-associated protein complex (DPC) composed of isoforms of syntrophin, dystroglycan, and dystrobrevin, which in turn associates with AQP water channels. Studies showing disturbed ion and water regulation in transgenic animals [49] confirm the importance of the polarized function at these key blood-CNS tissue interfaces.

8.9 Induction of BBB Properties in Brain Endothelium

8.8 Secretory Functions of Astrocytes

Astrocytes have been shown to express and release a number of agents capable of acting on other cells within the nervous system, including other macroglia, neurons, endothelial cells, and microglia. Substances released include neurotransmitter and modulator substances, cytokines, chemokines, and growth factors (Table 8.1); and the agents may have inductive effects (influencing the phenotype of the recipient cell), or act as shorter-term signaling molecules (see below). The majority of these studies have been on cells in culture, as it is very difficult to do the equivalent experiments in vivo or in brain slices. From the observed differences in morphology and cell-cell associations in vivo, one would predict that the perivascular and perineuronal cell processes of astrocytes could release different agents, but studies have generally not been done to test this idea. There are likely to be interesting differences in the effective extracellular compartment size between perisynaptic release sites (narrow extracellular spaces and synaptic clefts) [50] and perivascular release sites (facing the extracellular matrix surrounding the blood vessel, frequently with an expanded perivascular space). These differences in volume will give different degrees of dilution of the released agents; and the likely channeling of ISF flow in low-resistance perivascular spaces will be an additional factor, affecting both the dilution and the sphere of influence of the agent [44, 51]. These are some of the complexities likely to result from differences in the behavior of different astrocyte processes.

8.9 Induction of BBB Properties in Brain Endothelium

It is clear from the above discussion that the polarized properties of astrocytes play an important role at the blood-brain interface, particularly in relation to the physiology of ion and water homeostasis. The way in which close communication between astrocytes and endothelium leads to induction of BBB properties is covered in detail by Galla’s team [52] and Wolburg [53] (Chapters 16 and 4 in this volume). Here it is simply worth noting that these inductive influences depend on the polarized properties of both astrocytes and endothelium; and if either breaks down, for example by disruption of the anchoring proteins that stabilize the clustering of channels and transporters in particular membrane domains, then induction may be impaired [54–57]. Even before much was known about the mechanisms underlying the induction of barrier layers at specific sites within the CNS (in mammals: brain endothelium, choroid plexus epithelium, RPE, tanycytes, ependyma surrounding neurosecretory zones; in invertebrates: predominantly glial layers [17, 58]), it was proposed that induction could require the combined influence of factors from the blood side and from the brain side, with the final sites of barrier location being the cell layers giving the greatest separation of the two [17]. It was

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8 The Bipolar Astrocyte: Polarized Features of Astrocytic Glia Underlying Physiology Table 8.1 Substances secreted by astrocytes. Category

Substance

Reference

(A) Peptides/growth factors, proteins

Angiotensinogen

77, 78

GDNF BDNF NGF IGF1

79–84 81–85 82–86 87

ADNP

88

FGF1 (aFGF) FGF2 (bFGF) FLRG Endozepines

89 90 91 92

S100b GM-CSF

93–95 96

Clusterin APP

97 98

> 30 proteins (not all identified)

99

TGF-a TGF-b1 IL-1b, -6, -10, -12

TNFa MCP-1 IL-8 MIP3a/CCL20 Fractaline TenascinC

87, 100 87, 100–105 1b: 106, 6: 107, 108, 10: 109, 12p40: 110 111, 112 113 114, 115 116 117 118

MMP1, -2 MMP2, -3, -9 +tPA MMP9 TIMP-1

119 120 121 99, 119, 122

(B) Cytokines

(C) Chemokines

(D) Extracellular matrix proteins, modulators

Notes

Insulin-like growth factor +NAP, eight amino acids See also ApoE Follistatin-related gene Neuropeptide related to Octadecaneuropeptide (ODN) Granulocyte-macrophage colony-stimulating factor Sulphated glycoprotein Amyloid precursor protein Proteases, protease inhibitors, carrier proteins, antioxidants

Matrix metalloproteinases Tissue inhibitors of metalloproteinases

8.9 Induction of BBB Properties in Brain Endothelium Table 8.1 (continued) Category

Substance

Reference

(E) Sterols, lipidic substances

Cholesterol, 123 desmosterol, labosterol ApoA1 124 ApoE 89, 125–127 128 ApoJ PGD2 129 PGE2 87, 129, 130 Hydroxypregnane one 87

(F) Neurotransmitters, neuroactive agents

Arachidonic acid GABA

131 132

Glutamate ATP Nitric oxide

133 134–136 108

Notes

Cholesterol efflux to ApoA-1 ApoE synthesis, HDL secretion Prostaglandins 3-a-hydroxy-5-a-pregnane-20-one

proposed that when the tight junctions within an epithelial or endothelial layer became sufficiently occluding to restrict the diffusion of potential inducing proteins, then this cell layer would experience the greatest difference between apical and basal microenvironments, and would be the one in which barrier induction would subsequently be most effective. Moreover, as this layer tightened during development, it would automatically limit the access of plasma factors to underlying cells, confining the barrier site to a single cell layer. This is still a useful working hypothesis, which has been able to accommodate more recent cellular and molecular advances. Many of the factors released by astrocytes (Table 8.1) are able to induce specific features of the BBB phenotype in brain endothelial cells [59, 60]. In some cases, close contact between the basolateral surfaces of the endothelial and glial cells is more effective in BBB induction than astrocyte-conditioned medium, which may reflect the need to preserve the correct cell polarity for optimal interaction [61]. It is now clear that, in addition to the important role of tight junctions in the induction and polarization of CNS barrier properties, the extracellular matrix is a critical anchoring site mediating the polarizing influences at the endothelialastrocyte interface [53, 62] and in other polarized ependymoglial derivatives, such as the RPE [63]. New information is becoming available about regional differences in tight junctions, extracellular components, astrocyte polarity, and the inductive factors released by astrocytes and endothelium. This will aid our understanding of the more subtle aspects of induction, and disturbances in pathology, with possible insights leading to prospects for repair.

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8.10 Astrocyte-Endothelial Signaling

In addition to the longer-term inductive influences between astrocytes and endothelium discussed above, evidence exists for significant short-term cell-cell signaling between these cells [59, 64–66], with the polarized end feet of astrocytes on the capillary endothelium being the most likely key site for this signaling in vivo. Of the range of substances released by astrocytes (Table 8.1), a number are classic neurotransmitters and modulators, capable of acting not only on neurons but also on endothelium, pericytes, smooth muscle, and other perivascular cells [58]. For many of these agents, receptor mechanisms have been reported in brain endothelium [59, 67]. Evidence comes largely from cell culture studies in which molecular and functional examinations were used to identify the endothelial receptors and signal transduction pathways involved [59, 68]. Where it has been possible to use in situ or brain slice preparations, broadly similar results are found, although in some cases it appears that the intact system expresses a more limited receptor profile than seen in vitro (e.g. it has not been possible to detect functional P2Y2 receptors on capillary endothelium in slices, although these are well documented in cultured cells [59, 69]). The downstream consequences of activation of these receptors include modulation of a number of brain endothelial functions [70, 71], including permeability (tight junction modulation) [64, 67], transport [65, 66, 72], and enzymatic activity. In many of the cell culture studies, it has not been possible to determine whether the receptors are on the apical or basolateral membrane of the endothelium, although the latter would be expected for targets of astrocyte-released agents. However, in culture models preserving a relatively restricting paracellular (tight junctional) permeability, the efficacy of compounds applied to the apical and basal membranes can be compared [73–75]. In the in situ single pial microvessel preparation, agents are generally applied in the superfusate (basal side of endothelium), but can also be introduced via the lumen (apical side) [76]. Studies in brain slices offer the potential to examine whether signals released by astrocytes act only or directly on endothelial cells, or whether other cell types are involved [69].

8.11 Conclusion

This chapter has highlighted the way in which astrocyte polarity derives initially from polarity in the developing neural tube, but that this is modified thereafter by a combination of intrinsic and extrinsic influences that contribute to the bipolar and multipolar morphology of adult astrocytes and related ependymoglial derivatives in different brain regions. The importance of astrocyte polarity for function is emphasized, particularly in relation to induction of BBB features in

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9 Responsive Astrocytic Endfeet: the Role of AQP4 in BBB Development and Functioning Grazia P. Nicchia, Beatrice Nico, Laura M. A. Camassa, Maria G. Mola, Domenico Ribatti, David C. Spray, Alejandra Bosco, Maria Svelto, and Antonio Frigeri

9.1 Introduction

Maintenance of a constant internal osmotic environment is essential for normal cerebral activity. To this end, the blood-brain barrier (BBB) assumes an extremely important role in conjunction with astrocytes, which are involved in the regulation of water and ion homeostasis besides that of neurotransmitter metabolism and nutrient support of neurons. Astrocytes are very complex cells, capable of responding to a variety of external stimuli. One of their main functions is to control brain water and ionic homeostasis in order to optimize the interstitial space for synaptic transmission. To this purpose, the anatomy of astrocytes is characterized by an irregular cell body with abundant leaflet-like processes which: (1) come into contact with most synapses, (2) form endfeet on the brain surface to form the external “glia limitans”, and (3) form endfeet that completely envelop brain microvessels. Astrocytes are involved in the maintenance of the BBB, whose physiology is the product of collaboration between endothelial cells, pericytes, and astrocyte footprocesses. There are many transporters and channels at the level of perivascular astrocytic endfeet and they are responsible for solute transport in the CNS, since the BBB is impermeable to polar substances. These channels/transporters are responsible for important mechanisms, such as the transport of glucose, the main brain energy substrate, from the blood. In fact, astrocytic endfeet are enriched in glucose transporters and are capable of converting all or some of the glucose that enters their vascular endfeet to lactate before uptake by neurons. Moreover, astrocytes maintain the extracellular K+ concentration at levels which optimize neuronal function, by actively taking up K+ from the extracellular space and, at the level of astrocyte foot-processes, the excess intracellular K+ is shunted into the vascular system. In addition, chloride, sodium, and hydrogen ions are extruded into the vascular system across the cell membrane of perivascular astrocytes. Blood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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9 Responsive Astrocytic Endfeet: the Role of AQP4 in BBB Development and Functioning

9.2 Astrocyte Endfeet and BBB Maintenance

Astrocyte endfeet form part of the BBB, a complex glio-vascular system that controls the homeostasis of the central nervous system (CNS), preventing the non-specific passage of hydrophilic solutes between the blood and neuropil. Morphological and biochemical features of the brain capillaries, including endothelial tight junctions (TJs), the absence of endothelial fenestrations and vesicular transport, and the presence of membrane carriers, selectively regulate nutrient transport into the brain, thus ensuring neuronal protection (Betz and Goldstein 1986; Pardridge 1988; Reese and Karnowsky 1967). In addition to endothelial cells, the BBB is made up of pericytes, involved in angiogenetic processes and in the neuroimmune function (Balabanov et al. 1996; Hirschi and D’Amore 1997) embedded in the basement membrane. Endothelial TJs are the major structure responsible for restricting the paracellular escape of blood solutes across the cerebral endothelium, as well as for its polarity and high electrical resistance in vivo (Crone 1986; Madara et al. 1985, 1992). Structurally, TJs are composed of a complex belt-like zonula occludens close to the capillary lumen and integral membrane proteins (such as claudin, occludin, JAM proteins) and cytoplasmic accessory proteins (such as ZO-1, ZO-2, ZO-3, cingulin, 7H6) are involved in its molecular composition (Citi et al. 1988; Furuse et al. 1993, 1998; Jesaitis and Goodenough 1994; Martin-Padura et al. 1998; Stevenson et al. 1986; Zhong et al. 1994). Astrocytes form a continuous perivascular sheath closely apposed to the endothelial cells and pericytes, and become the main mediators between endothelial cells and neural tissue. Astrocytes are considered to be inducers of both the barrier and the permeability properties of the endothelium (Risau 1992). The earliest data on the inductive influence of astrocytes and the neural microenvironment on barrier features in the vessels came from studies performed on grafts of neural tissue in the coelomic cavity of different animals, which demonstrated that the newly formed vessels originating from the host displayed BBB characteristics (Stewart and Wiley 1981). Later, Janzer and Raff (1987), on injecting astrocytes into the anterior eye chamber of syngenic rats, demonstrated for the first time that a functional BBB was induced in non-neural endothelial cells, which vascularized the astrocytic aggregates. However, Krum and Rosenstein (1989) reported that astrocytes did not influence the barrier properties of the host endothelium in transplanted superior cervical ganglia and the same authors (1993) reported that no changes in endothelial permeability were detected after glial endfeet were damaged with a gliotoxin. These contrasting results might be due to different grafting techniques and graft vascularization analysis. Important findings on the relationship between astrocytes and endothelial cells derive from the coculture of endothelial cells and astrocytes or the use of conditioned media (CM). Even if caution in the interpretation of in vitro results must be used as reported by Holash et al. (1993), due to dedifferentiation of en-

9.2 Astrocyte Endfeet and BBB Maintenance

dothelial cells, it is commonly accepted that astrocytes exert an inductive action on the morphological, biochemical, and functional barrier features of endothelial cells. Brain endothelial cells cocultured with glial cells display a significant increase in TJ formation, together with an increase in enzymatic systems such as c-glutamyl transpeptidase (c-GT), Na+K+ATPase, alkaline phosphatase, and transporters such as neutral amino acids glucose (Beck et al. 1984; Cancilla and De Bault 1983; Meresse et al. 1989; Raub et al. 1992; Rubin et al. 1991; Tao Cheng et al. 1987). In addition, an up-regulation of the low-density lipoprotein (LDL) receptors and P-glycoprotein was also reported (Dehouck et al. 1994; Gaillard et al. 2000). Furthermore, endothelial cells cultured with astrocyte CM manifest an increase in TJ formation and electrical resistance, as well as in the expression of c-GT, ATPase, HT7, and neurothelin, suggesting the presence of soluble factors released by astrocytes, whose molecular composition has not yet been defined (Arthur et al. 1987; Lobrinus et al. 1992; Maxwell et al. 1987; Rubin et al. 1991). An important clue to the chemical nature of astrocyte-released soluble factors was given by Lee et al. (2003) who demonstrated that, after treatment of the cultured endothelial cells with src-suppressed C-kinase substrate (SseCKs)-CM, a factor stimulating the expression of angiopoietin-1 in astrocytes, TJ proteins increased and permeability to 3H-sucrose decreased. Moreover, various molecules such as transforming growth factor beta (TGF-b), fibroblast growth factor-2 (FGF-2) and interleukin-6 (Il-6) are able to induce some of the BBB properties induced by glial cells (Hoheisel et al. 1998; Sobue et al. 1999; Tran et al. 1999; Utsumi et al. 2000). BBB induction seems to be more effective in coculture where the glial processes come into contact with the basal endothelial surface. Hayashi et al. (1997) showed that non-neural endothelial cells developed barrier features when closely apposed to cocultured astrocytes, suggesting a key role of cell-cell contacts. Although a BBB induction was also observed with a neuronal membrane fraction (Tontch and Bauer 1991), astrocytes played the most important role in maintaining BBB properties. A particular role in the BBB is played by the perivascular astrocyte endfeet, which form a continuous perivascular layer and maintain cerebral homeostasis through a direct or indirect action on the endothelial cells, by regulating the ionic flux occurring during neuronal activity (Sun et al. 1995). The BBB also controls water transport, which is very important in CNS physiology, because it is involved with cerebro-spinal fluid (CSF) production, fluid transport across the endothelium and osmolality compensation in potassium siphoning (Nagelhus et al. 1998, 1999; Newman 1995; Walz and Hinks 1985). On the astrocytic endfeet membranes, a number of structures called orthogonal arrays of particles (OAPs) have been identified by means of a freeze-fracture technique (Dermietzel and Leibstein 1978; Neuhaus 1990). OAPs decrease on BBB damage (Wolburg et al. 1986), suggesting a role in the interaction between astrocytes and endothelial membrane. OAPs are associated with K+ siphoning (Newman 1995) and it has recently been demonstrated that a specific water channel protein,

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called Aquaporin-4 (AQP4; Ma et al. 1995; Rash et al. 1998), is involved in their molecular composition. Astrocytes seem to be influenced by their interaction with endothelial cells. OAPs increase after cocultivation with brain endothelial cells (Tao-Cheng et al. 1990); and their developmental expression takes place in parallel with TJ development (Nico et al. 1994). Moreover, the expression of AQP4 and glial fibrillary acidic protein (GFAP) at glial endfeet occurs in parallel with zonula occludens-1 (ZO-1) at endothelial TJs (Nico et al. 2004) and, in dystrophic mdx mice, open TJs are coupled with perivascular endfeet alterations (Nico et al. 2003). Astrocytes show a strong polarization in adult brains, where the BBB is structurally and functionally well developed, while they appear unpolarized in embryos when the BBB is absent or not completely differentiated (Bertossi et al. 1993; Wolburg 1995). In addition, the antioxidative activity of both astrocytes and endothelial cells in culture is higher than in monocultures of astrocytes (Schroeter et al. 1999) and it has been reported that endothelial cells release a factor which stimulates DNA synthesis in astrocytes (Estrada et al. 1990). Finally, it is noteworthy that astrocytes produce agrin, a heparan sulfate proteoglycan present in the subendothelial basal membrane of brain vessels and involved in BBB development (Barber and Lieth 1997; Warth et al. 2004). Thus, astrocytes may also contribute to BBB maintenance through the production of extracellular matrix components.

9.3 Astrocyte Endfeet and BBB Development

The development of CNS vasculature begins with perineural plexus vessels which penetrate the neuroectodermal layer and with new vessels originating from angiogenetic processes (Bar et al. 1995; Dermietzel and Krause 1991; Risau 1989, 1997; Risau and Wolburg 1990). These vessels radially penetrate the proliferative neuroectodermal tissue and connect with other vascular sprouts to give rise to a deep subventricular plexus in the brain anlage (Roncali et al. 1986). Brain angiogenesis is mediated by angiogenic cytokines such as fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factors (VEGFs) produced by the embryonic brain, which, by linking with their endothelial receptors, are strongly mitogenic for endothelial cells (Risau et al. 1988 a, b). While FGF-2 and its receptor expression do not correlate with BBB differentiation, VEGF and VEGF receptor expression (VEGFR-1, VEGFR-2) is coupled to brain vessel growth and differentiation. VEGF, in fact, is more highly expressed during embryonic life; and the newly formed vessels penetrate into the developing brain on the basis of the VEGF gradient produced by neuroepithelial cells (Risau 1997). Other angiogenic growth factors and their receptors are involved in brain vessel maturation, such as angiopoietin-1, -2 and their receptors Tie-1, -2 (Davis and Yancopoulos 1999), members of the transforming growth factor-b (TGFb) signaling pathways (such as TGFb1, ALK-1, SMAD5, SMAD6, endoglin), and

9.3 Astrocyte Endfeet and BBB Development

the ephrin family (for a review, see Gale and Yancopoulos 1999), as well as specific endothelial adhesion molecules, such as vascular endothelial (VE)-cadherin (Carmeliet and Collen 2000). The first embryonic brain vessels have no barrier properties. They are lined by a leaky endothelium with numerous vesicles and vacuoles, with no junction systems and are surrounded by neuroblast bodies and large perivascular spaces (Nico et al. 1999). During embryonic development, brain vessels progressively acquire barrier properties through progressive decreases in permeability and by expressing specific endothelial transporters and antigens (Dermietzel and Krause 1991; Dermietzel et al. 1992; Risau and Wolburg 1990). One of the most debated questions concerning BBB differentiation is the time-course of its maturation. By means of intravascular injection of permeability markers such as horseradish peroxidase (HRP), it has been demonstrated that, in some avian species, precocious brain development is already associated with a structurally and functionally developed BBB in embryonic life (Risau et al. 1986 a, b; Roncali et al. 1986; Wakai and Hirokawa 1978), whereas in rodents the BBB is completely differentiated and functioning only in post-natal life (Schulze and Firth 1992, 1993). The barrier vessel features are developmentally established by structural modifications involving both endothelial TJ and glial perivascular endfeet differentiation, as well as differential phenotypic antigen expression (Fig. 9.1 A, B). Freeze-fracture techniques and ultramicroscopic analysis on chick embryo brain and isolated microvessels have demonstrated that the spatio-temporal development of the complex networks of TJ strands occur in parallel with a complete sheet of the glial perivascular endfeet and mature OAPs (Fig. 9.1 C, D), together with basal membrane and pericyte recruitment (Nico et al. 1994, 1997). Moreover, along with a reduction in vessel permeability, BBB morphofunctional maturation is coupled with the expression of TJ proteins such as ZO-1 and glial endfeet proteins such as AQP4 and GFAP (Nico et al. 1999, 2001, 2004), confirming a mutual relationship between endothelial and glial cells in the induction of their membrane specialization. Recent studies demonstrated that the development of the glial endfeet expressing AQP4 is particularly important for controlling BBB differentiation and functioning. Immunogold electron microscopy (Fig. 9.1 D) demonstrated that this water channel protein is expressed in a polarized way on the glial membrane facing the vessels (Frigeri et al. 1995 a, b; Nielsen et al. 1997) and that its developmental expression occurs in parallel with OAP and TJ formation, and reduction of the perivascular embryonic spaces, depending upon water flux regulation (Nico et al. 2001). These results confirm the involvement of AQP4 in the molecular composition of OAPs and indicate a role of the glial endfeet in controlling the water flux balance at a BBB level, starting from embryonic life. Moreover, after BBB destruction obtained by treating embryos with LPS, glial swelling and brain edema occurs coupled with TJ opening and AQP4 reduction, showing a relation between glial endfeet polarity and BBB maintenance.

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Fig. 9.1 Ultrastructural features of the glial endfeet during BBB development in 14-day chick embryos (A, C) and 20-day embryos (B, D, E). (A) The microvessel wall is composed of endothelial cells with short TJs (arrow) and pericytes (p), which are discontinuously surrounded by isolated astroglial endfeet (arrowhead). Narrow perivascular spaces (asterisks) are recognizable. (B) A continuous layer of glial endfeet filled with glycogen granules (arrowhead) surrounds the basal lamina of a microvessel lined by a thin endothelium (e) and pericytes. (C) Replica from a fractured microvessel

showing the P-face of the plasma membrane of an astrocytic endfoot with small quadrangular OAPs (circles). (D) Replicas from fractured microvessels showing the E-faces of perivascular astroglial endfeet with a number of assembly pits with orthogonal symmetry (circles) corresponding to OAPs of the P-face. (E) Ultrastructural immunodetection of AQP4: numerous immunogold particles, singularly (arrowhead) or in clusters (arrow), decorate the plasma membranes of the astroglial endfeet (g) facing the vessel endothelium. Note the unlabeled endothelial cells (e) joined by a TJ (asterisk).

The close developmental relationship between glial endfeet and endothelial cells has been furthermore demonstrated in dystrophic mdx mice, a model of Duchenne muscular dystrophy, as well as in mdx embryos (Nico et al. 2003, 2004). We found that BBB alterations were developmentally established in mdx mouse brains (Nico et al. 2004). Biochemical maturation of the BBB takes place through changes in the expression of endothelial transporters and enzymatic systems (for reviews, see Betz and Goldstein 1986; Dermietzel and Krause 1991; Pardridge 1988; Risau and Wolburg 1990, 1991) which appear tightly coupled with astrocyte differentiation. In brain microvessels isolated from chick embryos, a high activity of alkaline phosphatase (ALKP) and neutral aminoacids (NAAs) and glucose transport levels was demonstrated before TJ formation (Nico et al. 1997). Moreover,

9.4 Astrocyte Endfeet and BBB Damage

in situ observations showed that endothelial junction differentiation was paralleled by a decrease in both endothelial pinocytosis and perivascular endfeet arrangement (Bertossi et al. 1992, 1993), further corroborating the notion that BBB structural and metabolic development is controlled by the glioneural environment. A number of other markers and proteins are also up- or down-regulated in their endothelial expression cells during BBB development (Risau et al. 1986a), such as the transferrin receptor (Kissel et al. 1998), the non-receptor tyrosine kinase Lyn (Achen et al. 1995), the HT7-antigen (Risau et al. 1986 b), the caveolar protein PV-1 (Stan et al. 1999), or the MECA 32 antigen (Hallmann et al. 1995). It is noteworthy that some endothelial transporters, such as P-glycoprotein, involved in the removal of lipophilic molecules and BBB differentiation (Qin and Sato 1995; Schinkel et al. 1994), or the glucose transporters (Glut-1), whose expression increases during development (Dermietzel et al. 1992), are also expressed in adults by perivascular glial endfeet (Morgello et al. 1995; Golden and Pardridge 2000), indicating a developmentally acquired astrocyte control in the pathways of glucose and other metabolites at BBB level.

9.4 Astrocyte Endfeet and BBB Damage

Disruption of the BBB is a consistent event occurring in the development of several CNS diseases, including demyelinating lesions in the course of relapsing multiple sclerosis, stroke, DMD, and mdx models, but also mechanical injuries (Ke et al. 2001, 2002), neurological insults (Vizuete et al. 1999), septic encephalopathy (Davies 2002), some brain tumors (Saadoun et al. 2002) where the BBB is poorly developed (Groothuis et al. 1991), and permanent ischemia or, more commonly, transient ischemia followed by reperfusion (Ke et al. 2001). In most cases, these pathological conditions are associated with an increase in microvascular permeability, vasogenic edema, swollen astrocyte endfeet, and BBB disruption. Astrocytes are activated very early in and around focal ischemic brain regions (Norenberg 1998). Reactive astrocytes are hypertrophic and hyperplastic, and can be identified by their increased expression of GFAP. Moreover, astrocyte swelling may negatively affect the already impaired blood circulation by causing vascular constriction. Nevertheless, endothelial cell swelling, thrombi formation, or vasoconstriction may also explain localized reductions in blood flow. Astrocytes play a significant role in host defense as well as in the pathogenesis of infectious and autoimmune diseases of the CNS. Astrocytes can be recruited to positively interact with T cells and seem to be involved in the downregulation of immune responses. They produce mediators that suppress MHC induction and reduce the activity of T cells. In fact, cocultures of macrophages with glial cells induce lower expression of adhesion molecule and MHC-II complexes, thus down-regulating their capacity to present antigens (Hailer et al.

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1998). Furthermore, astrocytes are capable of causing apoptosis in activated T cells that interact with locally presented (auto)antigen. Finally, astrocytes have been identified as one important source of chemokines for monocyte infiltration into the CNS (Weiss et al. 1998). Interaction between astrocytes and the immune system leads to an altered production of neurotoxins and neurotrophins by these cells, which intervene in the pathogenesis of HIV-associated dementia, multiple sclerosis, and Alzheimer disease; and a common feature in these diseases is the interaction of macrophages and astrocytes (Minagar et al. 2002). Moreover, stromal-derived factor-1 (SDF-1)-induced activation of CXCR4 on astrocytes leads to TNF secretion, thereby contributing to neuronal apoptosis (Bezzi et al. 2001). Astrocytomas are tumors composed predominantly of neoplastic astrocytes. In malignant astrocytomas, such as glioblastomas, the BBB is leaky, as visualized by the immunohistochemistry of plasma proteins using biopsy specimens (Seitz and Wechsler 1987), as well as by heterogeneous contrast enhancement upon neuroradiological examination (Roberts et al. 2001). Moreover, endothelial cells in tumor vessels show abnormal structural features, such as frequent fenestrations, prominent pinocytotic vesicles, lack of perivascular glial endfeet, as well as opening, loss, and/or abnormal morphology of TJs (Engelhard and Groothuis 1999). Experimental data have demonstrated that some astrocytoma cells implanted into rat brain become vascularized by leaky vessels, suggesting either a deficit in production of inductive factors by proliferating glioma cells, or an enhanced production of permeability factors that counteract the inductive effects (Bauer and Bauer 2000).

9.5 The Role of Aquaporins in BBB Maintenance and Brain Edema 9.5.1 AQPs Expression and Functional Roles

All the movements of solute/ions occurring at perivascular glial endfeet are associated with the movement of water. In the brain, as in other organs, water passes through plasma membranes via three distinct mechanisms (Agre et al. 2002; Verkman et al. 1996). The first is by facilitated diffusion and occurs through particular water channels called “aquaporins”, the second is by cotransport with organic or inorganic ions, and the third is by diffusion through the lipid bilayer. Brain neuropil, unlike epithelia in other organs, has a very complex three-dimensional structure, making it difficult to predict the direction and magnitude of water flux in physiological conditions (Nagelhus et al. 2004). Water transport has to be directly or indirectly coupled with homeostatic processes, such as glutamate uptake, potassium clearance, etc. Three aquaporins are expressed in brain: AQP1 is expressed in the choroid plexus epithelium (Nielsen et al. 1993), where it has been shown to

9.5 The Role of Aquaporins in BBB Maintenance and Brain Edema

Fig. 9.2 AQP4 expression in brain glial cells. (A) Schematic diagram showing the relationship between astrocytes (a), pial surface (ps) and blood vessels (bv). Astrocytes are irregularly shaped cells with many radiating processes that surround neurons (n) and the surface of blood vessels and extend to the ependymal and pial surface forming the glial limiting membrane. (B) AQP4 immunostaining (in red) is concentrated in astrocytic processes forming the

glia limitans and surrounding intracerebral capillaries. (C) High AQP4 expression is also found on the ependymal cells that are in contact with the CSF in the ventricular system. (D) Differential interference contrast image of the brain cortex showing AQP4 staining (red) along the perivascular glial endfeet. (E) Confocal analysis of AQP4 labeling at the perivascular glia membrane facing blood vessels.

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facilitate cerebrospinal fluid secretion (Oshio et al. 2004); AQP9 is found in a subset of astrocytes and in catecholaminergic neurons (Badaut et al. 2004); and the most expressed water channel in the CNS is AQP4 (Frigeri et al. 1995 a, b; Nielsen et al. 1997), principally by the astrocytes of the brain cortex (Fig. 9.2). AQP4 seems to be of primary importance in the brain’s handling of water (Agre et al. 2004; Amiry-Moghaddam et al. 2003 b; Manley et al. 2004; Papadopoulos et al. 2004 a b). Immunolocalization studies together with AQP4/GFAP double-staining experiments showed AQP4 expression strongly concentrated in astrocyte processes in direct contact with blood vessels (Frigeri et al. 1995 a, b; Nicchia et al. 2000). A confocal 3D reconstruction of a brain vessel stained with AQP4 antibodies shows strong AQP4 polarization where the glial endfeet are in contact with endothelial cells and demonstrates that the glial AQP4 positive endfeet completely envelop the vessel at the level of the BBB interface. High AQP4 expression has also been demonstrated at the subpial level, where the endfeet of numerous astrocytes form the glial limiting membrane (Frigeri et al. 1995 a, b), by the basolateral membrane of ependymal cells lining the intracerebral ventricles (Frigeri et al. 1995 a, b; Nielsen et al. 1997), and in spinal cord astrocytes (Fig. 9.3). Electron microscopy studies on ultrathin sections revealed that AQP4 gold particles appear to be numerous along the membranes of the perivascular glial endfeet facing the capillaries rather than those facing the neuropil (Fig. 9.4; Amiry-Moghaddam et al. 2003 b; Nicchia et al. 2000; Nico et al. 2001; Nielsen et al. 1997). AQP4 is also expressed in the glial

Fig. 9.3 AQP4 expression in ependymal cells and spinal cord. (A) AQP4 immunoperoxidase staining in the ependymal cell layer. Note that the choroid plexus (cp) cells are unstained. (B) Ependymal cells lining the ventricle display strong basolateral staining. (C, D) Dense AQP4 staining in the astrocytes of the spinal cord gray matter (gm). In white matter (wm), the staining is limited to fibrous astrocytes.


Fig. 9.4 Immunogold electron microscopy analysis of AQP4 expression in brain. AQP4 expression is mainly concentrated at the glial endfeet (arrowheads), while a few gold particles are detected on the neuronal side (arrows) of glial processes. RBC, red blood cell.

lamellae of the osmosensitive organs (Nielsen et al. 1997). Recently, it was reported that AQP4 is also expressed, at very low levels, by brain endothelial cells (Amiry-Moghaddam et al. 2004) and at non-endfeet membranes (Amiry-Moghaddam et al. 2003 b; Nielsen et al. 1997). Although in situ hybridization studies reported AQP4 expression in neurons (Venero et al. 1999, 2001, 2004), double-staining with the neuronal marker NFH (Neurofilament H) and electron microscopy analysis showed no AQP4 immunoreactivity in the bodies and cell processes of neuronal cells (Nagelhus et al. 1998; Nicchia et al. 2000; Nielsen et al. 1997). Strong AQP4 enrichment at the perivascular and subpial endfeet membranes suggests its cell biological function in governing homeostatic processes at the synaptic level (Nagelhus et al. 2004; Nicchia et al. 2004). For example, it has been suggested that AQP4 works in concert with Kir 4.1 to effect potassium buffering (Nagelhus et al. 2004). Indeed, in retinal Muller cells, AQP4 subcellular distribution matches that of Kir 4.1 (Nagelhus et al. 1999). Moreover, recent experiments performed with a-syntrophin null (a-syn–/–) mice support this hypothesis

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(Amiry-Moghaddam et al. 2003 b ). These a-syn–/– mice, which lack AQP4 (Hosaka et al. 2002; Neely et al. 2001) but have normal Kir 4.1 expression, showed a delayed K+ clearance after activation of synaptic areas in acute hippocampal slices (Amiry-Moghaddam et al. 2003 a). Thus, facilitation of K+ spatial buffering can be considered as a physiological role of AQP4. Moreover, since AQP4–/– mice show an increased seizure threshold (Binder et al. 2004), it was recently proposed that water flux through AQP4 contributes to the activity-dependent volume changes in extracellular space which affect neuronal excitability (Nagelhus et al. 2004).

9.5.2 The Role of AQP4 in Brain Edema

Important changes in water homeostasis are involved in a number of pathological processes, such as gliomas, brain edema, and head injury (Kimelberg and Ransom 1986). Astrocytic swelling is a common reaction to injury, with consequent impaired homeostasis mechanisms, uptake of neurotransmitters, and release of excitotoxic amino acids. Although the function of AQP4 in normal brain physiology is still unclear, its involvement in brain edema formation and resolution has been extensively demonstrated. Noteworthy in this field are the studies on AQP4 null mice showing that AQP4 deletion makes astrocytic foot processes more resistant to swelling after acute water intoxication and after ischemic stroke (Manley et al. 2000). Since AQP4, as well as other AQPs, allows a bidirectional water flow depending on the osmotic gradient (Amiry-Moghaddam et al. 2003 b; Meinild et al. 1998), it provides the major pathway for water entry and exit from the brain. In fact, recently, a role of AQP4 for vasogenic brain edema resolution was also demonstrated in AQP4 null mice. AQP4 would facilitate the removal of excess brain water (Papadopoulos et al. 2004 a) after continuous intracerebral fluid infusion. In order to study the function of AQP4 water channels in normal brain physiology, mouse models which lack AQP4 at the level of perivascular glia are available together with AQP4 null mice. These are the dystrophin-deficient mdx, mdx3cv and mdxbgeo mice, widely accepted animal models of Duchenne muscular dystrophy (DMD), and the a-syntrophin knockout mice.

9.5.2.1 DMD Animal Models The mdx and the mdx3cv mouse models have been extensively used by our group to study the role of altered water redistribution and thus of AQP4 in the pathogenesis of the neuromuscular abnormalities which characterize DMD. The mdx mouse lacks the dystrophin protein due to a genetic defect in the homologous region to the human DMD gene (Bulfield et al. 1984), but it manifests a less severe phenotype compared to the human pathology (Fig. 9.5). Dystrophin in skeletal muscle is a 427-kDa protein (full-length DP427) and it is


Fig. 9.5 Genomic organization of the dystrophin gene and the composition of the different dystrophin isoforms. (A) DMD gene, located in the short arm of the X chromosome (Xp21), is composed of 79 exons and occupies approximately 2.5 Mb. The gene encodes dystrophin and a family of N-terminal truncated isoforms that are produced by independent promoters (arrows). Three promoters drive the expression of full-length brain (B), muscle (M), and Purkinje (P) dystrophin isoforms that

differ only in the first few amino acids at the N-terminus. Activation of the other internal promoters produces short transcripts with a specific tissue distribution: Dp260 (retina), Dp140 (brain and kidney), Dp116 (Schwann cells) and Dp71 (ubiquitous). (B) Dystrophin proteins consist of four structural domains: the N-terminal actinbinding region with homology to a-actin, the rod domain with 24 spectrin-like repeats, the cysteine-rich domain and C-terminus domain.

proposed to link the extracellular matrix to the membrane cytoskeleton. In contrast, the brain contains DP427 plus two shorter dystrophin isoforms (DP140, DP71) produced by the activation of internal promoters (Gorecki et al. 1992). The mdx mouse model lacks only the full-length DP427, whereas the expression of DP140 and DP71 is unaffected. However, mdx3cv mice are the animal models of DMD in which all dystrophin isoforms are absent. Thus, since the main dystrophin isoform in the central nervous system is DP71 (Blake and Kroger 2000; Cox et al. 1993), which is expressed at the glial-vascular interface (Blake et al. 1999; Imamura and Ozawa 1998; Jancsik and Hajos 1999), the mdx3cv mouse is a better model for studying the brain alteration and cognitive impairments often associated with DMD. Our group demonstrated for the first time that AQP4 expression is strongly reduced (up to 90%) in mdx mouse sarcolemma (Frigeri et al. 1998, 2001) and

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Fig. 9.6 Immunogold electron microscopy of AQP4 in brain cortex of control, mdx and mdx3cv mice. (A) Control brain. Numerous AQP4 gold particles (arrows) decorate the glial membranes, separated by a basal lamina (asterisk) from the blood vessel. Magnification: ´ 20 000. (B) mdx brain from 1.5-year mdx mouse. A small number of gold particles are attached to the swollen glial endfoot (asterisk)

facing the blood vessels. Note the unlabeled glial membrane facing the neuropil (arrowheads). Magnification: ´ 20 000. (C, D) mdx3cv brain. Swollen glial endfeet (asterisk) enveloping two vessels are labeled by rare gold particles at the membrane facing the neuropil (C; ´ 12 000) or are devoid of gold particles (D; ´ 20 000) at the membranes facing the vessel and the neuropil. e, endothelial cell. (Reproduced from Nicchia et al. (2004), with permission).

to the same extent in DMD human muscle biopsies (Frigeri et al. 2002), suggesting that an altered osmotic balance may be seriously involved in the alteration of dystrophic muscles. Interestingly, the analysis of AQP4 expression in brain astrocyte foot processes of mdx mice revealed an age-related reduction of this water channel associated with swollen astrocyte processes (Frigeri et al. 2001) and BBB breakdown (Nico et al. 2003, 2004), suggesting a close relationship between BBB integrity and control of the water flux by astroglial cells (Nico et al. 2001). However, AQP4 reduction in mdx mice was significant only in older mice (Frigeri et al. 2001). To investigate whether the presence of other dystrophin isoforms in the mdx mouse, in particular DP71, may delay the reduction in AQP4 expression, we studied mdx3cv animals. The immunolabeling of perivascular astroglial endfeet membranes of a large number of mdx3cv vessels in several brain cortex areas displayed very reduced AQP4 staining (Fig. 9.6). Im-


munogold electron microscopy revealed that, in young mdx3cv mice, few AQP4gold particles were found at the glial endfeet surrounding the vessels, which appeared swollen (Fig. 9.6 C, D). Furthermore we noted that, in some vessels, a small number of gold particles were found at the level of glial membranes facing the neuropil (Fig. 9.6 D). This result indicates that AQP4 expression is more acutely affected in the mdx3cv than in the mdx mouse and that AQP4 reduction starts much earlier in the mdx3cv than in the mdx mouse. At 2 months of age, the mdx3cv mouse displayed a strong reduction in AQP4 immunolabeling, in conjunction with dilated glial perivascular spaces. The presence of DP71 in the brain seems to play a crucial role in the correct anchoring and stability of AQP4 at the glial perivascular endfeet. In normal mice, AQP4 expression at the perivascular glial endfeet determines rapid water movement, driven by the osmotic gradient in order to maintain brain water homeostasis (Amiry-Moghaddam et al. 2003 b, 2004; Frigeri et al. 2001; Manley et al. 2000; Nico et al. 2003; Vajda et al. 2002). The reduction in AQP4 associated with swollen glial processes found in mdx3cv indicates that water homeostasis is severely altered. In conclusion, our results suggest that DP71 plays a critical role in AQP4 expression at the astrocyte endfeet membrane adjacent to blood vessels in the cerebral cortex; and they strongly suggest that, as in mdx3cv, AQP4 reduction is involved in the brain modification occurring in DMD patients in which dystrophin mutation involves the DP71 isoform.

9.5.2.2 The a-Syntrophin Null Mice The a-syntrophin knockout mouse is the latest model available to study the role of AQP4 in brain physiology since, like mdx mice, it lacks AQP4 expression at perivascular level. a-Syntrophin is an adaptor protein, one of the dystrophin-associated proteins (DAPs), whose PSD95-disc large-ZO-1 (PDZ) domain is proposed to link the C-terminus of AQP4. In favor of this hypothesis, chemical cross-linking and coimmunoprecipitations from brain have demonstrated AQP4 in association with a complex, including dystrophin, beta-dystroglycan, and syntrophin (Neely et al. 2001). Moreover, studies on AQP4 expression in a-Syn–/– mice have demonstrated that AQP4 is absent at the level of skeletal muscle sarcolemma and that the polarized subcellular localization of AQP4 is reversed, being markedly reduced in astrocyte perivascular and subpial endfeet membranes, but present at higher than normal levels in membranes facing the neuropil (Neely et al. 2001). However, the need for a chemical crosslinker to obtain AQP4 and a-syntrophin co-immunoprecipitation and the demonstration that the expression and stability of AQP4 in the sarcolemma does not always decrease when a-syntrophin is strongly reduced (Frigeri et al. 2002) raises concerns about a direct interaction between AQP4 and a-syntrophin. Further biochemical studies are required to demonstrate the interaction between these two proteins. Likewise, in mdx mice, perivascular and subpial astroglial endfeet have been shown to be swollen in brains of a-Syn–/– mice compared with wild-type mice (Amiry-Moghaddam et al. 2003 b), suggesting that the clearance of water gener-

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ated by brain metabolism is reduced when AQP4 is absent or strongly reduced. In contrast, in pathological conditions, brain edema was attenuated after water intoxication in a-Syn–/– mice (Amiry-Moghaddam et al. 2004) as well as in AQP4 knock-out mice (Manley et al. 2000) and in mdx and mdx3cv mice (Frigeri et al. unpublished data) and also after transient cerebral ischemia in a-Syn–/– mice (Amiry-Moghaddam et al. 2003 b). However, the greater contribution of a-Syn–/– mice is related to studying the role of AQP4 in potassium buffering. In fact, since the expression of the inwardly rectifying K+ channel, Kir4.1, is grossly unmodified in these transgenics, they have been used to demonstrate that AQP4 and K+ channels work together to obtain an isosmotic clearance of K+ after neuronal activation. In fact, K+ clearance was prolonged up to 2-fold in a-Syn–/– mice compared with wild-type mice (Amiry-Moghaddam et al. 2003 a).

9.5.2.3 The Effect of Lipopolysaccharide on AQP4 Expression In order to clarify the role of AQP4 during conditions of BBB damage, we induced BBB disruption by LPS treatment in the optic tectum of 20-day chicken embryos in which the BBB is morphofunctionally well developed (Nico et al. 2001; Roncali et al. 1986). The BBB alterations induced by LPS during meningoencephalitis are characterized by a loss of endothelial tightness and an increase in vesicular content (Quagliarello et al. 1986). LPS-treated brains showed the presence of severe edema with swollen perivascular astrocytes, in agreement with the commonly accepted idea that glial swelling is a consequence of BBB damage. Immunogold and immunoperoxidase analysis revealed only a faint labeling of AQP4 protein on the astrocytic processes in the neuropil and around microvessels. The reduction of AQP4 protein expression on swollen glial endfoot associated with the damaged BBB further supports the close relationship between BBB function and water flux mediated by AQP4 perivascular expression and suggests that this water channel plays a key role in the genesis of brain edema.

9.5.2.4 AQP4 in Astrocytomas AQP4 is massively up-regulated in astrocytomas and this correlates with BBB opening assessed by contrast-enhanced computed tomographs (Saadoun et al. 2002). Warth et al. (2004) demonstrated that the redistribution of AQP4 in human glioblastomas correlates with a loss of agrin immunoreactivity. These authors postulated that agrin determines the polarity of astrocytes by binding alpha dystroglycan; and this polarity might be the precondition for the astrocytes’ ability to induce or maintain the BBB properties of brain endothelial cells.

9.6 AQP4 Expression in Astrocyte-Endothelial Cocultures


As discussed earlier, the specific structure of the BBB is based on the partnership of brain endothelial cells and astrocytes. Numerous in vivo studies have described interactive influences between astrocytes and vascular cells. In particular, several studies aimed to characterize the influence of astrocytes on endothelial cells (Arthur et al. 1987; Beuckmann and Galla 1998; Hurwitz et al. 1993; Janzer and Raff 1987; Stewart and Wiley 1981; Tao-Cheng et al. 1987; Tout et al. 1993), but the inductive influence of brain endothelium on astrocytes was also demonstrated (Beck et al. 1984; Estrada et al. 1990; Sperri et al. 1997; Wagner and Gardner 2000). Therefore, the maintenance of the BBB appears to depend on the continuous exchange of signals between astrocytes and endothelial cells; and disturbance of this exchange may be involved in several pathologies involving BBB dysfunction. In recent years, cocultures of these two cell types have been developed and used extensively as in vitro models to study aspects of barrier induction and modulation (Abbott 2002; Bauer and Bauer 2000; Hayashi et al. 1997; Krämer et al. 2001; Reinhart and Gloor 1997; Rubin et al. 1991). Astrocyte primary cultures constitutively express large amounts of AQP4 protein (Nicchia et al. 2000, 2003). Immunofluorescence analysis revealed that, although the expression of AQP4 appears to be clearly detectable on the plasma membrane with staining of the cell periphery, a significant amount of AQP4

Fig. 9.7 AQP4 expression in primary culture astrocytes. Double immunofluorescent confocal analysis of AQP4 (green) and GFAP (red) in purified astrocyte cultures grown on glass coverslips. Note the intense AQP4 staining at the cell periphery.

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A

Fig. 9.8 AQP4 expression analysis in rat astrocytes cocultured with endothelial cells (bEnd3) on glass coverslips. (A) Schematic diagram illustrating the coculture system. (B) AQP4 antibodies strongly stained the astrocyte processes in close contact with endothelial cells. (C) GFAP immunostaining of astrocyte endfeet surrounding a group of bEnd3 cells.

staining is observed in the cell cytoplasm (Fig. 9.7), indicating that the polarized expression of AQP4 observed in vivo is not maintained in purified astrocyte cultures. Thus, in brain the localization of AQP4 in the astrocytic processes close to the abluminal side of endothelial cells suggests that its position is chiefly determined by the BBB composition. To determine whether AQP4 polarized expression observed in brain could be reestablished in vitro and thus to dissect the mechanism that determines the anchoring of AQP4 in the astrocytic perivascular domain, we first analyzed the role of endothelial cells. To this purpose, astrocytes were cocultured with endothelial


Fig. 9.9 Electron microscopy analysis of astrocytes and endothelial cells in coculture. (A, B) Ultrathin sections of CE cocultured with astrocytes show thin layers of endothelial cells (asterisks) sealed by small TJs (arrowheads) and subtended by rounded processes of glial cells, containing glycogen granules and vesicles (A, arrow). Scale bars: A = 0.5 lm; B = 1.05 lm.

cells. For these studies, we used immortalized endothelial cells, bEnd3, originally derived from mouse brain capillaries (Williams et al. 1988, 1989) and freshly prepared rat astrocytes. Cells were cocultured in two different models: (1) by plating bEnd3 cells onto a confluent layer of astrocytes using the same medium as for the astrocyte cultures, or (2) by cultivating astrocytes on one surface of a porous membrane and endothelial cells on the opposite surface. In the first model, the two cell types are in direct contact with each other and after 7–14 days of coculture, the

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Fig. 9.10 Confocal microscopy analysis of AQP4 and GFAP protein distribution in astrocyte processes. Double immunostaining with AQP4 (red) and GFAP (green) antibodies. Note that the AQP4 staining is mainly distributed over the surface membrane.

presence of endothelial cells determined dramatic changes in the morphology of astrocytes (Yoder 2002), transforming them from a confluent flat monolayer into islands that were interconnected by elongated multicellular columns and thick processes (Fig. 9.8). Immunostaining with GFAP antibody confirmed that astrocytes were the cells forming those structures (Fig. 9.8 C). In many areas of the coculture preparation, endothelial cells were arranged to form capillary-like structures (Fig. 9.8 B) that were not seen when endothelial cells were grown alone or with other cell lines. Thus, a specific mutual induction occurs in cocultures. Indeed, other immortalized cell lines (glioma C6, RG2, neuronal GT1) were not able to induce similar morphological changes (Yoder 2002). Electron microscopy analysis revealed the presence of TJs between endothelial cells and thin astrocyte processes surrounding them (Fig. 9.9). The immunofluorescence analysis of AQP4 expression in the coculture system revealed an extraordinary redistribution of AQP4 in the astrocytes. In cocultured astrocytes, AQP4 staining appeared increased and was distinctly localized to those astrocytic processes in close contact to endothelial cells (Fig. 9.10). The staining was strongly associated with the plasma membrane, with little or no intracellular staining. Furthermore, the astrocyte somata also displayed membrane staining, but this was much fainter compared with that seen in the processes and was


Fig. 9.11 Analysis of AQP4 expression in rat astrocytes and endothelial cells (bEnd3) cocultured on 1-lm porous membranes. (A) Schematic diagram illustrating the coculture system. (B–D) Confocal microscopy analysis showing the presence of astrocyte processes on the endothelial side of the membrane. AQP4 antibodies specifically stained astrocyte processes (B), as demonstrated by GFAP immunostaining (C), in close proximity to bEnd3 cells. (D) Merged image of B, C.

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mainly comparable with levels seen in astrocytes alone. These results suggest that the presence of endothelial cells is necessary to induce the formation of astrocyte processes, which in turn are required for the localization of AQP4. In the second model (Fig. 9.11), endothelial cells were seeded on an insert membrane having 1.0-lm pores, on the opposite surface of which astrocytes had been directly seeded and grown. This system spatially separates the two cell types and thereby permits astrocytes to make contact with endothelial cells through their processes. Immunofluorescence analysis revealed strong membrane AQP4 expression in these GFAP-positive processes that traversed the pores and were in close contact with endothelial cells. No GFAP/AQP4 staining was detected on the opposite side of astrocytes when these cells were grown alone on the membrane insert, indicating that no cell processes passed through the pores. Furthermore, neuroblastoma cells (N2A) were not able to induce the formation of astrocyte processes. To our knowledge, this is the first evidence of a membrane protein whose polarized expression in vivo can be efficiently re-established in vitro. This induction of AQP4 polarization in astrocytic processes by close apposition to cocultured endothelial cells could be due either to a cellular differentiation process induced by endothelial cells (the result of a physical interaction between the two cell types), or to a diffusion of soluble signals. Preliminary coculture experiments performed using inserts with smaller pore sizes (0.2 lm) or astrocytes treated with conditioned medium did not reveal any phenotypic change, indicating that soluble molecules seem not to be involved in this phenomenon, whereas endothelial cells are required for the formation of cell processes. Future studies aim to analyze in detail the molecular aspect of this inductive effect on AQP4 polarity in coculture.

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Part III Hormonal and Enzymatic Control of Brain Vessels


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10 The Role of Fibroblast Growth Factor 2 in the Establishment and Maintenance of the Blood-Brain Barrier Bernhard Reuss

10.1 Introduction

The establishment and maintenance of a functional blood-brain barrier (BBB) is an important prerequisite for the special milieu of the central nervous system (CNS) and thus of proper brain function. In accordance with this, during alterations of the BBB during pathological injury and its restoration afterwards, it is important for the brain to cope with the special requirements for brain reorganization during wound healing. Fibroblast growth factors (FGFs) are amongst the earliest identified growth factors and have been shown to influence the growth and differentiation of brain microvascular endothelial cells, and thus to be involved in the establishment, maintenance, and restoration of the BBB. Especially recent findings on the role of FGF-2 (also known as basic FGF) for BBB formation and maintenance in vivo have brought this growth factor back onto the agenda of scientific interest. This chapter is intended to give an overview on past and present findings on the role of FGF-2 for BBB maintenance and function in the intact and lesioned CNS.

10.2 Role of FGF-2 in the Regulation of BBB Formation 10.2.1 Expression of FGF-2 in Astrocytes and Endothelial Cells of the Rodent Brain

Astrocytes and endothelial cells are both structural and regulatory partners for the formation and maintenance of the BBB; and thus an important question with regard to a role for FGF-2 in BBB-formation is whether FGF-2 and its appropriate receptors are expressed in either astrocytes and/or endothelial cells. In general during development of the CNS, a switch from a predominantly neuronal expression of FGF-2 during prenatal phases towards a primarily astrocytic Blood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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expression during postnatal phases has been described [1]. According to this at embryonic day (E)13 of rat development, FGF-2 is expressed in various neuronal precursor cell types. On E18, the cerebral cortex shows strong FGF-2 immunoreactivity and at the first postnatal day (PND1), FGF-2 is primarily located in neurons of the hippocampal formation. During PND4–PND6, astroglial expression of FGF2 begins, with the adult pattern of FGF-2 immunoreactivity in astrocytes from all brain regions being established at PND20. At that time neuronal FGF-2 expression is restricted to particular brain regions, such as the cingulate cortex and the hippocampal formation (CA2 field) [1]) This distribution seems to persist in the adult brain, where expression of FGF-2 has been demonstrated mainly in astrocytes [2], but also in special neuronal subpopulations [3, 4]. Besides such a predominant neural localization, at least during development, FGF-2 is also expressed in brain microvascular endothelial cells and therefore seems to be directly involved in endothelial cell differentiation during pre- and postnatal brain development. Support for this comes from a study by Schechter et al. [5], who demonstrated that, at the onset of vascularization during E15–E18 in the pituitary gland of the rat, the cytoplasm of the invading endothelial cells from immature capillaries shows intense immunoreactivity for FGF-2. Later (E19–E20), numbers of FGF-2-positive capillaries greatly decrease, with foci of released FGF-2 remaining still evident within the presumptive pericapillary spaces throughout gestation. In adult animals, however, capillary endothelial cells did not contain immunostainable FGF-2 in their cytoplasm [5]. Likewise a study by Marin [6] in the pineal gland demonstrated immunoreactivity for FGF2 in endothelial cells and perivascular spaces from PND20 onwards. This expression reached a maximum at the age of PND30–PND45, but unlike the pituitary gland, FGF-2 remains to be expressed also in later life [6]. This suggests that in several brain regions FGF-2 is only expressed during pre- and postnatal development, whereas in others FGF-2 expression continues to be present in the adult brain. With respect to expression of fibroblast growth factor receptors (FGFRs), in the adult brain expression of FGFR-1 mRNA and protein seems to be restricted to neurons, whereas FGFR-2 and -3 mRNAs are located preferentially in glial cells such as oligodendrocytes and astrocytes [7–9]. Again, FGFRs are also expressed in brain microvascular endothelial cells, as has been demonstrated for FGFR-1 in the median eminence of the pituitary gland of the rat [10]. Further evidence provided an in vitro study by Bastaki et al. [11] where FGFR-1 protein and mRNA were detected in a brain microvascular endothelial cell line.

10.2.2 Induction of BBB Properties in Endothelial Cells by Soluble Factors

Despite the above-mentioned localization of immunoreactive FGF-2 and FGFR1, brain endothelial cells are not capable to induce BBB properties on their own, but need to receive signals from the surrounding tissues (i.e. from astro-

10.2 Role of FGF-2 in the Regulation of BBB Formation

cytes). Such factors have indeed been found in astrocyte-conditioned medium and have been identified as hormones and protein growth factors [12, 13]. Thus, transforming growth factor(TGF)-b1 [14], glia-derived neurotrophic factor (GDNF [15, 16], interleukin (IL)-6, hydrocortisone [17], and FGF-2 [13, 18] have all been shown to induce at least some of the specific barrier properties of brain microvessels. With respect to FGF-2, Roux et al. [18] demonstrated in an immortalized cell line of rat brain endothelial cells (RBE4) that FGF-2 is able to induce RBE4 monolayers cultured on collagen-coated dishes to reorganize into three-dimensional tube-like structures. In addition, these endothelial tubes show increased activity of the BBB-associated enzymes c-glutamyl transpeptidase (c-GTP) and alkaline phosphatase, as compared with cell cultures growing only in two dimensions [18]. Likewise, as demonstrated by Sobue et al. [13], FGF-2 induces BBB properties in an immortalized endothelial cell line, as revealed by increased L-glucose permeability and alkaline phosphatase activity [13]. This effect could be blocked by the application of FGF-2 specific antiserum. However, the previous finding of Boado et al. [19], who demonstrated induction of the BBB-specific glucose transporter GLUT1 by FGF-2 in brain endothelial cells, could not be reproduced. There was also no observable induction of the expression of multidrug resistance genes which are normally present in brain endothelial cells [13].

10.2.3 Indirect Astrocyte Mediated Effects Seem to Play a Role in FGF-2-Dependent Changes in Endothelial Cell Differentiation

Although direct actions of FGF-2 on brain endothelial cell differentiation are of great importance, astroglial FGF-2 is also able to influence endothelial cell differentiation by indirect mechanisms. Thus, FGF-2 is able to elicit changes in astrocyte differentiation in an autocrine manner, which then indirectly affect endothelial cell differentiation and BBB properties. An important mediator for such indirect effects of FGF-2 on endothelial cell differentiation seems to be TGF-b1, which can be induced in astrocytes by FGF-2 and, upon its induction, modulates BBB-like differentiation of brain endothelial cells. This has been shown by Garcia et al. [20], who used a coculture model to demonstrate that local activation of TGF-b1 release from astrocytes is responsible for establishing at least some of the barrier properties in brain microvascular endothelial cells. Likewise Dohgu et al. [21] could show that TGF-b1 lowers the permeability of endothelial cells in culture and thus is involved in keeping the BBB function. Another feature of astrocytes that seems to have a major impact on the differentiation of brain microvascular endothelial cells and on BBB properties seems to be the intermediary filament proteins such as the glial fibrillary acidic protein (GFAP). In support of this, Pekny et al. [22] could demonstrate that astrocytes from wild-type mice are able (whereas astrocytes from GFAP-deficient mice are not able) to induce BBB properties in aortic endothelial cells in an in vitro co-

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culture model. Since FGF-2 is well known to influence the synthesis and phosphorylation of GFAP in astrocytes [23–25], this could then indirectly lead to changes in differentiation of brain microvascular endothelial cells by the abovementioned GFAP-dependent mechanism. This has indeed been demonstrated in a study by our group [26], where mice with a single or double deficiency in genes for FGF-2 and/or FGF-5 revealed reduced levels of GFAP in different brain regions. In addition, FGF-deficient animals showed also a leaky BBB as demonstrated by albumin extravasation as well as reduced levels of the tight junction proteins occludin and ZO-1 [26]. Whether this regulation also involves changes in astroglial expression and release of TGF-b1 will have to be clarified by additional experiments.

10.2.4 Involvement of FGF-2 in the Regulation of BBB Properties in the Pathologically Altered Brain

Another important aspect of FGF-2 function in the regulation of endothelial BBB properties might be also its implications for understanding the regulation of BBB permeability under pathological circumstances. Disturbed BBB permeability is a common feature of several disease conditions in the CNS, including ischemia, tumor growth, and demyelinating disorders [27–29]. Likewise, changes in FGF-2 expression under these conditions also suggest a role for FGF-2 in the regulation of BBB permeability under pathological circumstances. For example, after focal brain ischemia by middle cerebral artery occlusion (MCAO), expression of FGFR-1 mRNA is induced in capillary endothelium in the corpus callosum and internal capsule [30]. Together with increased astroglial FGF-2 expression found in the brains of rats suffering from MCAO dependent ischemia [31], this suggests that stimulation of FGFR-positive capillary endothelial cells by astroglia-derived FGF-2 could play a role in the restoration of blood flow and the reorganization of brain vascularization after ischemic damage. This view is further supported by the fact that intracerebral xenografts of mouse bone marrow cells in adult rats facilitate the restoration of cerebral blood flow and BBB by the release of FGF-2 [32]. Similar conclusions are drawn from an in vitro study of Brown et al. [33], which demonstrated that conditioned medium from the C6 glioma cell line can induce BBB properties in brain microvessel endothelial cells and protect them against hypoxia-induced BBB breakdown. This protective effect is accompanied by significantly higher levels of FGF-2 in C6-conditioned medium and increased expression of the tight junction protein claudin-1 in endothelial cells [33]. Likewise, inhibition of vascularization during tumor growth of C6 and 9L glioma cell lines by angiostatin is accompanied by decreased expression of vascular endothelial growth factor (VEGF) and increased expression of FGF-2 mRNA, suggesting that in this case proliferation of endothelial cells and thus vasculariza-

10.3 Future Perspectives

Fig. 10.1 Direct and indirect modes of action are thought to play a role in FGF-2-dependent regulation of microvascular endothelial cell differentiation.

tion might be reduced in parallel with a higher rate of terminal differentiation of endothelial cells [34]. A group of disorders where FGF-2-dependent regeneration of BBB properties in endothelial cells could be involved are the demyelinating diseases, an important feature of which is the breakdown of the BBB (for a review, see [35]). In accordance with this, in an experimental in vivo demyelination/remyelination model, levels of FGF-2 and of its receptors are greatly increased, with their peak expression being at the initial stage of remyelination [36].

10.3 Future Perspectives

In conclusion, FGF-2 is one of the key regulators for the formation of the BBB during brain development and for BBB maintenance in the adult brain (for an overview, see Fig. 10.1). In addition, it seems to be even more important for the restoration of proper BBB functions after brain injury and a lack in FGF-2 seems to be the cause of BBB leakiness in solid brain tumors. Of special significance therefore is the indirect impact of FGF-2 on endothelial cell functions by regulating the interactions of the latter with astroglial perivascular endfeet. Together these properties of FGF-2 make it an interesting candidate for pharmacological intervention in the regulation of blood flow and revascularization after brain injury or BBB permeability during the treatment of solid brain tumors.

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11 Cytokines Interact with the Blood-Brain Barrier Weihong Pan, Shulin Xiang, Hong Tu, and Abba J. Kastin

11.1 Introduction

After peripheral production, cytokines exert diverse effects on CNS functions. These CNS effects can occur in several ways. Cytokines can cross the bloodbrain barrier (BBB) directly, reach the CNS by retrograde axonal transport, act on circumventricular organs, or activate secondary mediators without themselves entering the brain and spinal cord. This chapter will focus on the current knowledge from pharmacokinetic studies of cytokine permeation across the BBB and on the intracellular events of cytokine trafficking in cerebral microvessel endothelial cells. Representative cytokines discussed include the interleukins, tumor necrosis factor a, leukemia inhibitory factor, epidermal growth factor, basic fibroblast growth factor, transforming growth factor, and some neurotrophins. These cytokines have pronounced neuroendocrine and trophic effects. We will also discuss some cytokines and chemokines that alter the morphology and intracellular signaling pathways of the cerebral microvessel endothelial cells. They are implicated in CNS inflammation, tumor metastasis, cerebral amyloid angiopathy, and various etiologies of stroke. As shown in disease models such as spinal cord injury, experimental autoimmune encephalomyelitis, and stroke, the regulation of the interactions between the cytokines and the BBB play important roles in CNS physiology and pathology. The BBB interfaces the parenchyma of brain and spinal cord and its supplying capillary vessels. The structural components are microvessel endothelial cells, pericytes, astrocytic endfeet, and extracellular matrix. The endothelial cells are joined by tight junctions, lined by a continuous basement membrane, and have reduced pinocytic vesicles and increased metabolic and enzymatic activity. These structural features are involved in the relative impermeability of the BBB to large proteins in the blood circulation. However, there are many instances in which cytokines produced in the periphery have CNS effects, and most of such actions are mediated by the BBB. In this review, we will discuss two principal ways by which cytokines interact with the BBB: (a) their transport or transcytoBlood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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sis across the endothelial cells that are the structural backbone of the BBB and (b) their actions on these endothelial cells which result in altered endothelial function, cytotoxicity, or cell proliferation.

11.2 Identification of the Phenomena 11.2.1 Cytokines That Cross the BBB by Specific Transport Systems Interleukins Interleukin-1a and -b are among the proinflammatory cytokines that have a saturable influx transport system [8, 12]. In their studies, Banks et al. [8, 12] reported an influx transfer constant of 0.25–0.43 ll g–1 min–1 for interleukin-1a and 0.47 ll g–1 min–1 for interleukin-1b. The initial volume of distribution was 20.1 ll g–1 and 16.5 ll g–1, respectively. The relative stability of the interleukins in the circulating blood and in the brain has been shown by high performance liquid chromatography (HPLC) and acid precipitation. Although there is influx transport, shown by self-inhibition, there is no saturability of brainblood efflux. This vectorial passage indicates that interleukin-1 is an important mediator in the communication between the CNS and the periphery. Regional differences in the rate of transport illustrates that the transport system for interleukin-1a is physiologically relevant to CNS function. Interleukin1a gets into the hypothalamus more rapidly than other parts and there is selective uptake in the posterior division of the septum [8, 9]. The region-specific uptake is probably related to the suppression of feeding behavior and catabolic metabolism induced by interleukin-1a. The circumventricular organs (CVOs) account for less than 5% of the total brain uptake of interleukin-1a, and permeation into the CVOs can still be saturable [74]. Since interleukin-1 is pyrogenic, the reduced brain permeation of interleukin-1b in aged animals may partially explain the diminished fever response in the elderly [47]. Interleukin-6 also has a saturable influx transport system, shared by human and mouse interleukin-6 and flagged mouse interleukin-6, but not by interleukin-1 or tumor necrosis factor a (TNFa) [10]. After intravenous delivery by a bolus injection, intact interleukin-6 is recovered from the cerebrospinal fluid (CSF) at 10 min and 30 min. The lack of saturable efflux indicates that this transport system is unidirectional. Tumor Necrosis Factor a Although large doses of TNFa might disrupt the BBB and increase paracellular permeability of the BBB, TNFa can cross the BBB by a saturable influx transport system. Trace amounts of 125I-TNFa can be detected by HPLC in blood, brain homogenate, and CSF 30 min after intravenous delivery, without increasing the permeability of co-administered 99mTc-albumin [31]. Most of the injected TNFa enters brain and spinal cord parenchyma. The regional differences in uptake are such that the spinal cord has higher permeation than the

11.2 Identification of the Phenomena

brain [53]. In the spinal cord, the cervical and lumbar segments have higher permeability reflected by a greater volume of distribution and faster influx rate. In the brain, the hypothalamus and occipital cortex appear to take up TNFa significantly faster than the rest of the brain [11]. The influx of TNFa is blocked by pre-incubation with a soluble receptor against the p75 receptor [13], and it is absent in double TNF receptor knockout mice [65]. The regulation of such receptor-mediated transport of TNFa will be further discussed later in this review. Leukemia Inhibitory Factor and Cilliary Neurotrophic Factor Cilliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) cross the BBB by independent saturable transport systems [68, 69]. Both have moderately fast influx rates (0.46 ll g–1 min–1 for CNTF, 0.41 ll g–1 min–1 for LIF). Although the two cytokines have one shared receptor subunit, gp130, there is no known cross-inhibition. The high affinity receptor gp190 is apparently involved in the transport of LIF, since a blocking antibody specifically reduces the influx transfer constant of LIF in both mouse studies and cultured brain endothelial cells [56]. Insulin-Like Growth Factor 1 The availability of insulin-like growth factor 1 (IGF-1) in the blood circulation for crossing the BBB is significantly influenced by IGF binding proteins (IGFBPs). To deliver sufficient amounts of IGF-1 to the CNS compartment, one has to first saturate the binding sites of IGFBPs. Regardless, there is a saturable influx transport system for IGF-1 at the BBB [61], which is at least partially shared with that for insulin [71]. The cross-inhibition of transport between IGF-1 and insulin indicates a receptor-mediated mechanism. The possible beneficial effects of IGF-1 include reduction of neurodegeneration and amelioration of autoimmune damage to the CNS [40, 43]. Thus, manipulation of the transport system and design of IGF-1 variants that can cross the BBB easily should be important goals for future research. Epidermal Growth Factor Like IGF-1, epidermal growth factor (EGF) is a trophic factor both in the periphery and the CNS. EGF has a saturable transport system from blood to brain that does not seem to involve its receptor, and there is no efflux transport [60]. A potential problem for the delivery of EGF and IGF1 as therapeutic agents for CNS pathology is that both may promote the growth of tumors such as prostate cancer.

11.2.2 Cytokines That Permeate the BBB by Simple Diffusion Glial Cell-Derived Neurotrophic Factor Glial cell-derived neurotrophic factor (GDNF) has relatively fast degradation in the blood circulation; such instability may be explained by its endogenous production in the CNS. There is no saturable transport for GDNF, but there is simple diffusion across the BBB to some

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extent. Unlike GDNF, many other neurotrophins, such as nerve growth factor and neurotrophin-3, can be transported from blood into the CNS [54, 75]. Interleukin 10 Interleukin 10 is more stable in blood than many cytokines, with no apparent degradation at 30 min after intravenous injection. It also has relatively high lipid solubility compared with other proteins of similar size (160 amino acids), having an octanol buffer partition coefficient of 0.183 ± 0.004 and hydrogen bonding of 0.965. Regardless, there is very limited simple diffusion of interleukin 10 from blood to brain in the mouse, and its apical-basolateral flux is similar to that of the paracellular permeability marker albumin in cultured endothelial cells [38]. Chemokines Of the CXCR family of chemokines, cytokine-induced neutrophil chemoattractant-1 (CINC1) has limited passage cross the BBB by simple diffusion but shows significant binding to the apical surface of cerebral microvessel endothelial cells [62]. Interleukin 8 is relatively more stable but also crosses the BBB only by simple diffusion, although its influx transfer constant is significantly lower than that of CINC1 [66]. The interactions between chemokines and the CXCR family of receptors are important in leukocyte migration and tumor metastasis to CNS parenchyma. The macrophage inflammatory proteins MIP-1a and MIP-1b are very stable in blood because of their polymerization. They have a high apparent volume of distribution in brain; however, their high binding to the luminal surface of cerebral blood vessels did not lead to endocytosis and penetration across the BBB during the period of study [7].

11.2.3 Cytokines That Have Known Effects on Endothelial Cells

TNFa affects cytoskeletal arrangement and tight junction protein distribution, which in turn increases paracellular permeability at high doses [72]. Platelet-activating factor is involved in the re-arrangement of cytoskeletal organization induced by TNFa [19]. Depending on the concentration and species of endothelial cell, TNFa may stimulate nitric oxide production or activation of the phospholipase A2 pathway [27]. More importantly, TNFa induces the expression of intracellular adhesion molecule-1 [44, 46], vascular cell adhesion molecule-1, and E-selectin [14, 24]. The interactions of TNFa and the endothelial cells could promote both the transport and paracellular permeation of TNFa and other molecules, including the 4.7-kDa peptide urocortin [52]. The interactions are also important in mediating inflammatory responses and brain metastasis of cancer cells. Endothelial cells express both p55 and p75 receptors for TNFa. In the human endothelial cell line ECV304 and other cells, p55 is predominantly located in the trans-Golgi network [37]. This subcellular localization is related to a C-terminal sequence of 23 amino acids that contains an acid patch and a dileucine mo-

11.2 Identification of the Phenomena

Fig. 11.1 Effects of filipin and chlorpromazine on the internalization of TNFa in TM-BBB4 cells. TM-BBB4 cells were incubated with filipin (5 lg ml–1) or chlorpromazine (25 lg ml–1) for 30 min at 37 8C. The cells were incubated with filipin or chlorpromazine in the presence of 125I-TNFa at 37 8C for an additional 30 min. Surface binding was determined by an acid wash procedure. The percent of 125I-TNFa internalized was determined after cell lysis. There was a significant decrease in the percent of 125I-TNFa internalized in the presence of either filipin (P < 0.05) or chlorpromazine (P < 0.01).

tif which interact with membrane traffic adaptor proteins [82]. Upon phosphorylation, such as by the p42 mitogen-activated protein kinase (MAPK), p55 translocates to intracellular tubular structures associated with the endoplasmic reticulum [21]. The trafficking of the p75 receptor is less well known. The differential locations of the two subtypes of receptors may not only be important in cell signaling but also in receptor-mediated transport of TNFa across the BBB. How does intracellular signal transduction then affect the endocytosis and intracellular trafficking of TNFa? In an immortalized mouse brain microvessel endothelial cell line, TM-BBB4, endocytosis of 125I-TNFa is mediated both by clathrin-coated pits and caveolin. As shown in Fig. 11.1, both chlorpromazine (an inhibitor of the clathrin-mediated pathway) and filipin (an inhibitor of caveolinmediated endocytosis) significantly reduced the internalization of TNFa at 30 min. Although it is known that p42 MAPK is involved in the translocation of the p55 receptor, inhibitors for MAPK, including PD98059 and SB203850, had no effect on endocytosis, the initial and rate-limiting step for transport of 125ITNFa (Fig. 11.2).

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Fig. 11.2 Effect of MAPK inhibitors on the internalization of TNFa in TMBBB4 cells. TM-BBB4 cells were cultured with PD98059 (25 lM) U126 (1 lM), and SB203850 (1 lM) for 1 h at 37 8C. 125I-TNFa was added and the cells were incubated at 37 8C for an additional 30 min. The surface binding was determined by an acid wash step; and the percent of internalized 125I-TNFa was not significantly different among the groups.

Transforming growth factor a (TGFa) is a potent angiogenic factor [25]. TGFa can promote the interactions of tumor cells with the endothelial cells composing the BBB, facilitate cancer cell colonization and invasion across the BBB, and stimulate tumor growth inside the CNS [49]. Thus, TGFa plays a significant role in tumor metastasis to the brain. It may also induce proliferation of endothelial cells after mild injury and thereby contribute to the angiopathy of intracranial vessels [79]. Therefore, although TGFa does not have a saturable transport system for crossing the BBB [70], its interactions with the BBB are implicated in various CNS pathologies. Chemokines and their receptors on the apical surface of endothelial cells are involved in inflammation, tumor metastasis, and endothelial proliferation. In particular, monocyte chemoattractant protein-1 (MCP-1) acts on the CCR2 receptor on endothelial cells, causes redistribution and down-regulation of tight junction proteins, and increases the permeability of the BBB, both in vivo and in vitro [80]. The expression of MCP-1 is up-regulated by proinflammatory cytokines including TNFa, IL-1b, and interferon c [33].

11.3 Mechanisms of Cytokine Interactions with the BBB

11.3 Mechanisms of Cytokine Interactions with the BBB 11.3.1 Endocytosis of Cytokines by the Apical Surface of Endothelial Cells

The membrane events that lead to transport of a cytokine from blood to the CNS could involve carrier-mediated transport, receptor-mediated endocytosis, and adsorptive endocytosis. There are many factors that determine the nature of the endocytic pathways, such as the size of the endocytic vesicle, the cargo (ligands, receptors, lipids), and the mechanisms of vesicle formation. Clathrin, caveolin, or non-clathrin, non-caveolin-mediated endocytosis has been reviewed in detail by experts in cell biology [20]. Transport of TNFa across the BBB involves both p55 and p75 receptors. This was first shown in studies with TNF receptor knockout mice [65]. The absence of either the p55 or the p75 receptor reduces the influx transfer constant of 125ITNFa, an effect significant in the brain but less pronounced in the spinal cord. When both receptors are absent, however, transport of TNFa is completely abolished. The involvement of both receptors is also evident in transwell studies on primary mouse brain microvessel endothelial cells from double receptor knockout mice [59]. In addition to TNFa, compounds such as LIF, IGF-1, and the adipokine leptin also can be transported by receptor-mediated endocytosis. Adsorptive endocytosis has been shown for ebiratide, an analog of adrenocorticotropic hormone. Ebiratide is a basic peptide with an isoelectric point of 10.0. Its blood-brain influx is saturable, energy-dependent, and inhibitable by other polycationic peptides and endocytosis inhibitors such as protamine and dansylcadaverine [83]. The occurrence of adsorptive endocytosis implies that the positively charged ebiratide moieties interact with the negatively charged cell surface in a specific manner. The binding affinity is lower than what would be expected for receptor-mediated endocytosis, but the transport capacity is substantially higher. However, another basic polypeptide, basic fibroblast growth factor (bFGF), crosses the BBB with an intermediate capacity by specific binding to heparin sulfate proteoglycan; and its endocytosis is neither receptor- nor adsorptionmediated [22].

11.3.2 Intracellular Trafficking Pathways

In many types of polarized cells, the binding of a protein or peptide ligand to the cell surface receptor is followed by alterations in membrane dynamics. The ligand-receptor complex is concentrated in microdomains that are rich in either clathrin or caveolae. After a series of events to recruit adaptor molecules, membrane invagination leads to vesicular transport of the ligand-receptor complex to

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early endosomes. There are two likely fates. In one situation, the complex can be guided to lysosomes where increased acidity (lower pH) causes dissociation of the ligand from the receptor with recycling of the receptor back to the cell surface and degradation of the ligand. Alternatively, the complex can be trafficked to common endosomes and then elsewhere for a secretory pathway. It can also be directed to the Golgi complex and eventually to the secretory pathway. Regardless, the apical to basolateral migration of a cytokine may follow a tortuous path. Not all cytokine ligands can be transported across the endothelial cells, and probably even fewer pass the four-dimensional structure of the BBB. It has been shown in recent years that monoubiquination plays an important role in determining the fate of an endocytosed protein [36]. Ubiquitin is an abundant and highly conserved peptide of 76 amino acids. It is added posttranslationally to lysine residues on proteins and is recognized by the cellular machinery that targets them for proteolysis. Cytokine receptors, which are membrane proteins, might be susceptible to ubiquination, generating signals for regulated internalization and intracellular sorting.

11.3.3 Signal Transduction in Endothelial Cells

The fate of a cytokine inside the endothelial cells is largely dependent on its transport cargo. For instance, specific amino acid sequences in the cytoplasmic domain of a receptor can be the sorting signal to determine the site of degradation for a cytokine. How signal transduction triggered by a cytokine then affects its trafficking pattern is not clear.

11.3.4 Involvement of Other Cells Composing the BBB

In addition to endothelial cells, the BBB also contains pericytes, astrocytic endfeet, and neuronal components, and it is surrounded by extracellular matrix. The regulatory roles of elements of the BBB other than endothelial cells have been reviewed elsewhere [6].

11.4 Regulation of the Interactions of Cytokines with the BBB Circadian Rhythm TNFa is a cytokine that can affect circadian rhythms in both physiological and pathological conditions. The uptake of TNFa by the spinal cord, but not by the brain and peripheral muscle, shows a multiphasic response that is not simply sinusoidal [57]. Circadian changes in BBB permeation are also seen for OSIP, an enkephalin analog [77], interleukin-1a, TNFa [57], and leptin [63].

11.5 Stroke and Other Vasculopathy

Feeding Status, Obesity, and Cachexia Our preliminary study shows that the serum concentrations of TNFa and interleukin-6 are in the pg ml–1 range, whereas that for interleukin-1b is not detectable. Fasting and obesity do not seem to significantly affect the concentrations of these cytokines, whereas cachexia is known to be related to increased blood concentrations of proinflammatory cytokines [2]. Whether altered feeding status affects BBB permeation of cytokines is not clear. Spinal Cord Injury and Brain Trauma Traumatic injury to the CNS induces cytokine production in a time- and region-dependent manner in situ; and there are increased levels of certain cytokines in the blood circulation. We have shown that both spinal cord injury and mild traumatic brain injury increase the specific transport of TNFa independently of barrier disruption [64, 67, 73]. Cytokines can also alter the expression of adhesion molecules such as E-selectin after trauma [24]. Primary and Metastatic Tumors, Inflammation, and Autoimmune Diseases Intracranial neoplasms, inflammation, and autoimmune disorders are related to a partially disrupted BBB and altered expression of cytokines and their receptors in the brain. Regarding direct permeation of cytokines across the BBB in such situations, we have shown that transport of TNFa is upregulated after experimental autoimmune encephalomyelitis [55].

11.5 Stroke and Other Vasculopathy

We have been focusing on the role of TNFa in stroke. Real-time PCR analysis of gene expression profiles in rats after transient middle cerebral artery occlusion (MCAO) shows that TNFa expression is increased 3 h after stroke and lasts for 24 h in the hemisphere ipsilateral to occlusion. This is accompanied by changes in the mRNA for interleukin-1, interleukin-6, E-selectin, and intercellular adhesion molecule-1 (ICAM-1), in the immunoreactivity for activated nuclear factor (NF)-6B, and in the infiltration of inflammatory cells [15]. After permanent MCAO, TNFa expression is increased not only in neurons, but also in astrocytes, microglia, choroid plexus, endothelial cells, infiltrating polymorphonuclear leukocytes, and probably pericytes. Upregulation of the p55 receptor for TNFa is present at 6 h and precedes that of the p75 receptor, which occurs at 24 h [18]. Both injurious and beneficial roles have been proposed for TNFa in the pathogenesis of cerebral ischemia [48]. On the one hand, blockade of TNFa actions by dimeric type I soluble TNF receptor (a TNFa binding protein) in BALB/C mice [50], anti-TNFa antibody Pl14, and a synthesis inhibitor CNI-1493 in Lewis rats [48] all reduce infarct volume after permanent MCAO. On the other hand, TNFa pretreatment induces neuroprotection against permanent MCAO in BALB/C mice

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with reduction of infarct size and CD11b-positive neutrophils and macrophages [51], whereas mice lacking the p55 TNFa receptor (R1) have a greater infarct volume than wild-type and p75 receptor knockout mice after MCAO and sustain more damage of CA3 hippocampal neurons by kainic acid excitotoxicity [29]. One possible explanation of these paradoxical results is that the TNFa blocking reagents were administered peripherally, whereas TNFa pre-treatment was given by the intracisternal route. The time of administration probably accounts for the different actions of TNFa, as the two types of studies most likely involve activation of different cellular targets. Regardless, TNFa is crucially involved in the initiation, progression, and regeneration processes after stroke, and in the development of tolerance to ischemia [32, 45]. As stroke alters BBB function and dynamically changes communication of the CNS with the periphery, transport of vascular TNFa across the BBB after stroke probably contributes to the effects of TNF in the CNS. We have shown that transport of TNFa is significantly increased 1 week after tMCAO, and this increased function is mediated by upregulation of the p55 and p75 receptors in endothelial cells [58]. Interactions of TNFa with cerebral endothelial cells affect not only its own transport but also the paracellular permeability of the BBB. TNFa changes cytoskeleton organization, tight junction protein expression, and production of serine proteases, including tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), and matrix metalloproteinases (MMPs) [23, 78, 81]. These serine proteases are involved in BBB disruption, tissue remodeling, and neural plasticity [1, 3, 17, 86]. Exogenous tPA as a thrombolytic agent binds to the LDL-receptor related protein (LRP) at the BBB and can induce opening of the BBB [84]. TNFa also induces cerebral endothelial cell expression of genes involved in cell adhesion, chemotaxis, apoptosis, neuroprotection, transcriptional regulation, etc. [28]. Thus, TNFa affects stroke progression and recovery at the BBB level.

11.6 Neurodegenerative Disorders

Amyotrophic lateral sclerosis (ALS) is a progressive and devastating disorder affecting motor neurons. There has been evidence that the general permeability of the BBB is increased in ALS patients, as indicated by increased serum and CSF levels of albumin, and increased CSF total protein and immunoglobulin [41, 42]. TNFa might exacerbate neurodegeneration by potentiating the release of the excitatory neurotransmitter glutamate [16]. In ALS patients, TNFa concentrations in blood are increased [76]. The linkage of TNFa and neuroinflammation is evident in an animal model of familial ALS by gain-of-function mutations in Cu, Zn superoxide dismutase (SOD1). TNFa mRNA expression appears in the spinal cord of young SOD1 mice before the development of symptoms of motor weakness or

11.7 Summary

significant motor neuron loss and correlates with the severity of the disease [26]. The SOD1 mice have upregulation of the p55 TNFa receptor in the presymptomatic stage, supporting the concept that inflammation in the CNS occurs relatively early and precedes the onset of frank paralysis and accumulation of bulk protein oxidative damage [35]. Microarray analysis of cDNA from the spinal cord of SOD1 mice also shows that the mRNA expression of TNFa increases eight-fold and TNFa immunoreactivity is increased in microglia and motor neurons of the spinal cord. Along with other factors, it seems that the inflammatory process mediated by TNFa directly participates in neuronal death [85]. Further study in the spinal cord of SOD1 mice shows that TNFa, transforming growth factor b, interleukin 10, some chemokines, and other proinflammatory cytokines like interferon c (IFNc) and interleukin-1, -2, and -6 are also increased. However, nitrite efflux assays in cultured Walker EOC-20 microglia cells show that TNFa causes the maximal cellular response. The results suggest that TNFa is the principal driver for neuroinflammation, with its effects potentiated by co-stimulating cytokines and chemokines [34]. It is yet to be determined whether TNFa transport across the BBB is altered in ALS, and whether modulation of such transport can modify the course of neurodegeneration. LIF is a neurotrophic cytokine to motor neurons, although the systemic sideeffect of cachexia is seen when it is administered in large doses. In a pilot study, LIF appeared to reduce the loss of motor neurons in the lumbar spinal cord of SOD1 mice after daily intraperitoneal injection for 6 weeks [4]. Although a follow-up study from the same research group failed to reproduce the results, LIF seemed to have a positive effect on locomotor behavioral tests by delaying the onset and reducing the severity of symptoms [5]. The lack of a substantial and sustained benefit in this study could be related to multiple factors including sex difference, generation variability, sensitivity of the tests, as well as the dose, duration, and route of administration. In addition, a transport defect of LIF crossing the BBB may explain why the effects of LIF are not sustained after 3 months of age. In normal mice, a saturable transport system at the BBB is responsible for blood-brain and blood-spinal cord entry of LIF [68]. In the presence of dysregulation of the BBB, peripherally administered LIF may not reach its CNS targets effectively. This could also be part of the explanation for the inconsistent results with LIF in clinical trials of ALS patients [30, 39].

11.7 Summary

The BBB is a target of action for many cytokines; and it also serves as a dynamic regulatory interface in communications between the CNS and the rest of the body. A limited number of cytokines can permeate the BBB by way of saturable transport systems. The details of intracellular trafficking and the regulation of transport in pathophysiological states are the subjects of ongoing studies. The BBB provides a potential site for modulation of the effects of cytokines on the CNS.

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Acknowledgment

This work was supported by the NIH (NS45751, NS46528, DK54880, AA12865).

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1997, Tumor necrosis factor a: a neuromodulator in the CNS, Neurosci. Biobehav. Rev. 21, 603–613. Pan, W., Zhang, L., Liao, J., Csernus, B., Kastin, A. J. 2003, Selective increase in TNFa permeation across the blood-spinal cord barrier after SCI, J. Neuroimmunol. 134, 111–117. Plotkin, S. R., Banks, W. A., Kastin, A. J. 1996, Comparison of saturable transport and extracellular pathways in the passage of interleukin-1a across the blood-brain barrier, J. Neuroimmunol. 67, 41–47. Poduslo, J. F., Curran, G. L. 1996, Permeability at the blood-brain and bloodnerve barriers of the neurotrophic factors: NGF, CNTF, NT-3, BDNF, Mol. Brain Res. 36, 280–286. Poloni, M., Facchetti, D., Mai, R., Micheli, A., Agnoletti, L., Francolini, G., Mora, G., Camana, C., Mazzini, L., Bachetti, T. 2000, Circulating levels of tumor necrosis factor-alpha and its soluble receptors are increased in the blood of patients with amyotrophic lateral sclerosis, Neurosci. Lett. 287, 214. Ramge, P., Kreuter, J., Lemmer, B. 1999, Circadian phase-dependent antinociceptive reaction in mice determined by the hot-plate test and the tail-flick test after intravenous injection of dalargin-loaded nanoparticles, Chronobiol. Int. 16, 767–777. Rosenberg, G. A. 2002, Matrix metalloproteinases in neuroinflammation, Glia 39, 279–291. Sidawy, A. N., Mitchell, M. E., Neville, R. F. 1998, Peptide growth factors and signal transduction, Semin. Vasc. Surg. 11, 149–155. Stamatovic, S. M., Shakui, P., Keep, R. F., Moore, B. B., Kunkel, S. L., Van Rooijen, N., Andjelkovic, A. V. 2005, Monocyte chemoattractant protein-1 regulation of blood-brain barrier permeability, J. Cereb. Blood Flow Metab. (in press). Stolphen, A. H., Guinan, E. C., Fiers, W., Pober, J. S. 1986, Recombinant tumor necrosis factor and immune interferon act singly and in combination to reorganize human vascular endothelial cell monolayers, Am. J. Pathol. 123, 16–24. Storey, H., Stewart, A., Vandenabeele, P., Luzio, J. P. 2002, The p55 tumor necrosis factor receptor TNFR1 contains a trans-Golgi network localization signal in the C-terminal region of its cytoplasmic tail, Biochem. J. 366, 15–22. Terasaki, T., Takakuwa, S., Saheki, A., Moritani, S., Shimura, T., Tabata, S., Tsuji, A. 1992, Absorptive-mediated endocytosis of an adrenocorticotropic hormone (ACTH) analogue, ebiratide, into the blood-brain barrier: studies with monolayers of primary cultured bovine brain capillary endothelial cells, Pharm. Res. 9, 529–534. Yepes, M., Sandkvist, M., Moore, E. G., Bugge, T. H., Strickland, D. K., Lawrence, D. A. 2003, Tissue-type plasminogen activator induces opening of the blood-brain barrier via the LDL receptor-related protein, J. Clin. Invest. 112, 1533–1540.

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Doyu, M., Sobue, G. 2002, Differential expression of inflammation- and apoptosis-related genes in spinal cords of a mutant SOD1 transgenic mouse model of familial amyotrophic lateral sclerosis, J. Neurochem. 80, 158–167. 86 Zhuo, M., Holtzman, D. M., Li, Y., Osaka, H., DeMaro, J., Jacquin, M., Bu, G. 2000, Role of tissue plasminogen activator receptor LRP in hippocampal long-term potentiation, J. Neurosci. 20, 542–549.

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12 Insulin and the Blood-Brain Barrier William A. Banks and Wee Shiong Lim

12.1 Introduction

Insulin has profound effects on the CNS. These effects are mediated through receptors localized throughout the CNS. As the brain produces little or no insulin, the source of cerebral insulin must be, ultimately, the pancreas. Thus, insulin secreted into the blood by the pancreas must cross the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) to enter the CNS. This chapter will review the evidence which shows that insulin enters the CNS by a saturable transport mechanism. This transport is crucial to the exertion of insulin’s full physiological effects. The characteristics of this transport are not static but affected by physiological changes and pathological conditions. The characteristics of the saturable transporter directly influence the ability of insulin to affect the CNS. Both the capillary endothelia and choroid plexus contain insulin receptors, that is, cell surface binding proteins coupled to intracellular machinery. Insulin thus has effects on the physiology of the cells which comprise the BBB. One such effect is to alter or modulate some of the BBB transport systems which could, in turn, affect the function of the CNS cells behind the BBB. Thus, the BBB regulates the access of insulin to the CNS and insulin, in turn, regulates some aspects of BBB function. This chapter will examine the complex interactions between the BBB and insulin as they are currently understood.

12.1.1 Early Studies

Early studies indicated that insulin did not cross the BBB. Three studies conducted in 1954–1962 had injected radioactive insulin into the blood stream of rats, dogs, and humans but detected little or no radioactivity in brain or CSF. Blood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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Measurements using the rat adipose tissue bioassay also did not detect significant amounts of insulin in the CSF. The first of these studies was by Elgee et al. and was conducted in 1954. Their objective was to investigate the biodistribution of insulin after intravenous (iv) injection; and brain was only one of many tissues studied. The study carefully considered many potential pitfalls. For example, the study used the power of radioactively labeled insulin to track biodistribution, it showed that radioactive insulin retained biological activity, it used acid precipitation to distinguish radioactive degradation products from intact insulin, it used fasted animals, and it used a ratio method (radioactivity in tissue vs radioactivity in whole body) to decrease statistical variation. The study was also comparative in that, while most of the studies were conducted in rats, a parallel arm of the study injected radioactive insulin into four “agonal” patients at 25 min to 8 h before their death. The study also suffered from a common problem of biodistribution studies which happen to include brain. Peripheral tissues, especially kidney and liver, took up the majority of radioactive insulin. In comparison, brain uptake seemed negligible. In rats, only erythrocytes took up less insulin than brain. In humans, little or no insulin was found in the CSF or brain. This finding fit well with the idea that the brain was not an insulin-sensitive tissue. Haugaard et al. in the same year published a complementary study. They injected insulin labeled with either radioactive sulfur or radioactive iodine into rats or dogs. Again, these were pioneering studies addressing the fate of intravenous insulin and its distribution among the major tissues of the body. In one set of studies which included brain, they co-injected radioactive albumin as a measure of vascular space. Brain was the only tissue which did not obviously take up insulin. They concluded that little or no insulin entered the brain and, like Elgee et al., noted this was consistent with the view that “metabolism of the brain is . . . independent of the presence of insulin.” The above findings were reinforced a few years later by the study of Mahon et al. They assayed human CSF for insulin-like activity, using the rat adipose tissue technique. They estimated that they should be able to determine insulin levels as low as 3% of the concentration in serum with this method. However, they found no evidence for insulin in the CSF. They also injected iv radioactive insulin into humans and CSF obtained from the lumbar region. Radioactivity in blood and CSF was acid-precipitated to distinguish the degradation products. They conducted their study to 90 min, by which time they felt so much insulin had been degraded that the study should not be extended. Acid-precipitable radioactivity was only found in CSF in the 90-min sample. The counts were extremely low and in all cases were much less than 1% of the serum counts. Despite these negative results, the authors made the very conservative conclusion that the results did “not exclude the possibility that insulin is active in the central nervous system.” In retrospect, the study of Mahon et al. is the easiest to critique. The bioassay available to them was crude by the standards which would be set in the next few years by the work of Berson and Yalow. Taking CSF from the lumbar region

12.1 Introduction

was a distinct, but almost always necessary, disadvantage in humans. That same year, Grundy [1] would show that the CSF of the spine and the brain do not mix well. As such, the levels of peptides in brain CSF often correlate with peptide levels in blood, whereas levels in spinal CSF and blood usually do not [2]. At this point, it might have seemed that any insulin which was in the CNS was negligible. But the idea that insulin might affect the CNS was being reconsidered. Furthermore, studies had found that insulin injected into the brain produced hypoglycemia. This raised the possibility that insulin might act within the CNS. The invention of the radioimmunoassay by Berson and Yalow introduced a much more sensitive way to measure insulin. In 1967, Margolis and Altszuler used the radioimmunoassay to revisit the question of whether insulin could cross the BBB [3]. They found a low amount of insulin (3 lU ml–1) in the CSF of dogs, or about 27% of blood levels. They then infused large amounts of insulin and glucose intravenously for up to 6 h. CSF levels of insulin also increased, although with a lag before reaching a steady state. The higher infusion rates produced higher levels of insulin in the CSF, but the increase was not proportionate. Approximations made from their figure 2 indicate that the CSF/serum ratio was about 0.27 (i.e. 27% of serum values) at the baseline serum value of 11 lU ml–1, 0.07 at serum levels of 275 lU ml–1, and 0.03 at serum levels of 1600 lU ml–1. Thus, Margolis and Alszuler [3] concluded that insulin crossed the blood-CSF barrier by a saturable transport system rather than by passive diffusion. Margolis and Altszuler followed up their study to show that insulin injected into the CNS could also enter the blood. Although they argued from their results that the efflux of insulin was not saturable but by the mechanism of CSF reabsorption (bulk flow), they also concluded that insulin was able to cross the

Fig. 12.1 Increase in publications for a search combining (CNS or brain) and insulin, using the Library of Congress. Intervals are for 5 years (for example, 1960 refers to the interval 1956–1960). For the first interval (1951–1960), there were no publications. For the last full 5-year interval (1996–2000), 1413 publications were located.

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Fig. 12.2 Reanalysis of the data of Greco et al. (1970) by plotting serum and CSF values for baseline condition (open circles “normal”, in upper and lower panels) and for hyperinsulinemic condition (closed circles, lower panel). Results show a significant relation for the baseline condition (n = 21, r = 0.510, P < 0.05). Combined results for baseline condition and hyperinsulinemic condition show a nonlinear relation consistent with saturable transport of insulin across the BBB.

BBB in both directions, that is, from blood to CNS and from CSF to blood. Thirty years later, the implications of such bidirectional movement would be debated not only for insulin, but also for peptides, cytokines, feeding hormones, and many other substances. Ironically, the efflux of insulin from CSF to blood provided an explanation for how CNS insulin induced hypoglycemia. Rather than being mediated through central mechanisms, the insulin was merely entering blood to work through peripheral insulin receptors. However, within a few years, new studies would begin to redefine the role of insulin in the CNS. Figure 12.1 shows the increase in CNS insulin publications over the past 50 years from 1950 until 2000.

12.1 Introduction

The idea that insulin could cross the BBB was reinforced by the study of Greco et al., who revisited the question of insulin in the CSF of man, this time using a radioimmunoassay. They examined diabetic and nondiabetic patients and split their analysis into two groups: those with normal levels of serum insulin and those with high levels of serum insulin. They found insulin in the CSF of all patients. In those patients with high serum insulin, the insulin in the CSF was also elevated, although not proportionately so. On this basis, they suggested that insulin crossed the BBB by an active mechanism as opposed to passive diffusion. Figure 12.2 reanalyzes the data from their 1970 paper by plotting serum and CSF values. Interestingly, a statistically significant linear correlation exists between the serum and CSF insulin values for patients with normal insulin levels and a nonlinear model fits all data. This is consistent with their conclusion of a saturable transport system for insulin from blood to CSF. With these three studies, it might seem that the question of insulin passage across the BBB was once again settled, this time in favor of insulin crossing the BBB. But studies would occur throughout the 1970s and 1980s at prominent laboratories and published in prestigious journals that concluded that insulin was not able to cross the BBB [4, 5]. It fell to the next generation of studies to further define the relation between insulin and the BBB.

12.1.2 Debates Related to the Question of Permeability of the BBB to Insulin

In retrospect, three prominent issues seem to have impacted on the question of whether insulin could cross the BBB. Although these issues did not directly deal with the physical transfer of insulin across the BBB, they did frame the context in which the BBB studies were interpreted. These issues were: 1. Does the CNS make insulin? If the CNS could make its own insulin, then why would it need to import insulin from the outside? If the CNS did not make insulin, then did not the mere presence of insulin in the CSF and brain tissue indicate passage across the blood-brain and blood-CSF barriers? 2. Can proteins/peptides cross the BBB? The BBB was defined in the late 19th century by its ability to restrict dyes which bound tightly to albumin. This concept was rigorously generalized by many to all substances with peptide bonds, even the small, biologically active peptides. But the 1970s and 1980s found increasing proof that small peptides could cross in both the CNS-blood and blood-CNS directions. This suggested a form of communication between the CNS and peripheral tissues mediated through the BBB, an idea which resonated well with the ideas of Margolis and Altszuler in 1967. How much the ongoing work with small peptides influenced the insulin field is unclear, as insulin with its double-stranded structure does not meet the usual definitions of peptides.

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3. Does the brain need insulin? Whether insulin was needed for glucose uptake by the CNS was a hotly debated topic. If insulin was not needed to either drive insulin across the BBB or into neurons, what useful role could it serve within the CNS? A slowly emerging counter-argument, foreshadowed by Margolis and Altszuler in their second paper, was that insulin had actions in addition to glucose uptake. Could insulin have roles within the CNS other than glucose regulation? Each of these questions have aspects or derivative questions which are very much a topic of investigation. Full answers to them and a review of what aspects are currently being investigated are beyond the scope of this chapter. However, a summary of current thinking as it affects this review are given here: 1. Does the CNS make insulin? It seems generally agreed that the vast majority of insulin in the CNS of the healthy adult originates from the pancreas. If the CNS does make insulin, it seems to be localized regionally or to specific circumstances. However, as reviewed by Plata-Salaman [6], several studies have found a paradoxic increase in brain insulin levels in animals whose pancreatic insulin output had been greatly diminished by streptozotocin. 2. Can peptides/proteins cross the BBB? Many peptides and proteins have been shown to cross from CNS to blood or from blood to CNS. Such passage has been shown in some cases to provide the mechanism by which a peptide or protein originating in one compartment (e.g. the periphery) influences the function in the other compartment (e.g. the CNS). 3. Does the brain need insulin? The general consensus is currently that, whereas the CNS is largely “insulin-independent”, defined as not requiring insulin for utilization of glucose, it is insulin-sensitive, i.e. insulin has many effects on brain function. Insulin receptors were found to occur throughout the CNS, including the cells which comprise the BBB. These receptors mediate several actions, some of which are discussed further below.

12.1.3 Does Insulin Cross the BBB? The Middle Years

The first 20 years of investigation of whether insulin crossed the BBB had produced mixed results. The earliest studies had failed to find insulin in the CSF or to show that radioactive insulin injected intravenously could be recovered from the CNS. Furthermore, it seemed likely that the brain did not require insulin for use of glucose. A second set of papers, however, used the newly invented, highly sensitive radioimmunoassay to show that insulin was in the CSF. Furthermore, it seemed that a correlation existed between the level of insulin in the CSF and the level in blood. Such a correlation was interpreted as evidence that CSF insulin was derived from serum.

12.1 Introduction

The studies of the next 20 years (1970s–1980s) largely confirmed these suspicions. Two major lines of investigation in particular reinforced the idea that insulin could cross the BBB: (a) evidence of insulin in the CNS, (b) the demonstration of insulin receptors and binding sites within the CNS and at the BBB. A seminal paper in the debate about insulin transport was published in 1977 [7]. This paper clearly showed that a correlation existed between the CSF and serum levels of insulin in dogs. A bolus injection of 0.2 lU of insulin increased serum levels by over 150-fold. CSF levels of insulin were also increased, but only by 2- to 3-fold. The same amount of insulin delivered as an infusion that elevated serum insulin levels by 4-fold also increased CSF levels of insulin. However, the increase in CSF was only about 2-fold and it was delayed 15– 30 min after the start of the infusion. Bolus injections and infusions of glucose increased the serum but not CSF levels of insulin. However, the increases in serum insulin with glucose administration were much smaller and more transient than the increases seen with insulin administration. When the CSF levels were regressed against serum levels for all data (basal levels and steady-state levels after glucose and insulin infusion), a correlation existed. However, this paper also pointed out difficulties that were complicating not only studies of insulin transport but also studies of other peptides and regulatory proteins. The results showed that CSF increases were small compared with serum levels, especially when studied by bolus injection. An apparent delay occurred between rises in serum and CSF levels, raising the question of which values of serum should be compared to which values of CSF. Comparison of the areas under the curve offered some degree of solution but also presented its own set of difficulties. These and related issues of how to analyze results for peptides and regulatory proteins were not fully resolved until the application of the methods of Patlak, Blasberg, and Fenstermacher [8, 9] several years later. Subsequent studies refined the experimental approach and demonstrated correlations between CSF and serum insulin levels in rats, man, and baboons [10– 12]. Additionally, insulin was clearly demonstrated to be present in brain tissue [13]. The delay in the increase in CSF levels was explained as the need for the insulin to move through a compartment between the CSF and serum, possibly the brain interstitial fluid [10]. Finally, the nonlinear association between CSF and serum levels of insulin was clearly demonstrated [14]. This nonlinearity could be readily explained if serum insulin entered the CNS by way of a saturable transporter. However, not all studies agreed that insulin crossed the BBB or even that there was a correlation between CNS and serum levels of insulin [4, 5, 15, 16]. Instead, the presence of insulin in the CSF and brain was interpreted as representing CNS insulin production. Especially difficult to ignore were a series of experiments previously reviewed [6], in which animals with streptozotocininduced insulinopenia had unaltered levels of insulin in the brain. Whether insulin was produced within the CNS in quantities sufficient to explain the levels found in CSF and CNS continued to complicate the question of insulin transport into the 1990s [6]. If no insulin was produced within the CNS, then CNS

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insulin must be derived from the periphery and so had crossed the BBB. To the degree that the CNS could produce insulin, the role of BBB transport was unclear. At first it may seem that a strong correlation between CSF and serum levels demonstrates that the substance is crossing the BBB. However, correlations between CSF and serum constitute a weak or presumptive proof of BBB transport. This is because it could be that release from the CNS and peripheral sources are responding independently, but to the same stimulus. Such has been shown to be the case for arginine vasopressin [17]. An important parallel set of studies involved the demonstration of insulin binding sites at both the choroid plexus and the brain endothelium [18–21]. Although the presence of such binding sites was not controversial, what those binding sites represented was. Alternate interpretations, sometimes by the same laboratory, were that these sites represented g-coupled receptors, transporters, or enzymes. Alternate positions held, sometimes by the same laboratory, were that insulin did not cross the BBB at all, crossed the endothelial barrier but not the choroid plexus, crossed at the circumventricular organs, crossed the choroid plexus but not the endothelial barrier, or crossed at both the endothelial barrier and the choroid plexus [20, 22, 23]. In retrospect, the BBB has binding sites which fulfill all three of these functions. Brain endothelial cells, for example, can degrade insulin [21, 24], contains g protein-coupled receptors which affect brain endothelial cell function [25–28] and can transport insulin across the BBB. These binding sites are not static, but vary during development and with other conditions [29, 30], consistent with the pathophysiological alterations in insulin transport noted below.

12.1.4 Insulin, the BBB, and Pathophysiology: The Past Decade

As it became increasingly clear that the CNS production of insulin was minimal, at least in healthy adults [11, 31], and increasingly accepted that a portion of the binding sites at the BBB represented transporters, work began to focus less on whether insulin crossed the BBB and more on what the pathophysiological implications of such transport represented. Nevertheless, these latter studies revisited the issue of insulin transport and largely confirmed with a different set of techniques that insulin clearly crosses the BBB. Before examining the pathophysiological work, it may be worth examining the new work showing that insulin crosses the BBB. Two variations on the older work of examining transport confirmed that insulin can cross the BBB. The first variation used radioactively labeled insulin in pharmacokinetic studies. The second variation used species-specific immunoassays to distinguish between exogenous and endogenous insulin. Both of these approaches solved the old problem of whether CNS insulin was made by the brain or transported from the periphery, using the same logic. Since the rodent

12.1 Introduction

brain could not make radioactive insulin, any radioactive insulin recovered from brain after its peripheral injection must have crossed the BBB. Similarly, any human insulin appearing in rodent brain after its peripheral injection must have crossed the BBB. Poduslo et al. [32] in 1994 examined the ability of radioactively labeled insulin to cross the BBB and also the blood-nerve barrier. They compared the transport rate of insulin with that of several other proteins, including albumin. Insulin was transported across the BBB at a rate of 1.36–0.94 ll g–1 min–1. This rate was well over 100-fold faster than the rate at which albumin leaked into the brain. These rates matched almost exactly the rates found by us several years later [33]. Human insulin injected intravenously appeared in the mouse brain [34]. Species-specific ELISAs capable of distinguishing between human and rodent insulin confirmed that the CNS insulin was of peripheral, exogenous origin. These studies showed a nonlinear relation between serum and brain levels of human insulin, consistent with saturable transport across the BBB. Fitting the data to a one-site hyperbola model showed that the transporter had reached half of its maximal capacity at a blood level of about 3.5 ng ml–1 and was about 30% saturated at levels as low as 1.0 ng ml–1. Blood glucose was not affected by serum insulin levels below 1 ng ml–1. This shows that substantial amounts of insulin are being transported into the brain at serum levels of insulin too low to cause hypoglycemia. This, in turn, suggests that insulin transport and CNS effects of insulin are important at physiologic, nonhypoglycemic levels of serum insulin. A study conducted with radioactive insulin also showed that insulin transport occurs at physiological levels [33]. This study found insulin transport rates to range over 0.87–1.70 ll g–1 min–1, rates which agreed very well with those found previously by Poduslo et al. [32]. A dose of 0.1 lg per mouse of unlabeled insulin inhibited transport across the BBB of radioactive insulin by about 50% without producing hypoglycemia. Again, this showed that substantial amounts of insulin are transported across the BBB at the physiologic, euglycemic levels of insulin. These studies clearly showed, regardless of the ability of the CNS to produce insulin, that insulin crossed the BBB by a saturable mechanism. They showed that insulin transport was substantial, far exceeding the amount which could be accounted for by the residual leakiness of the BBB [32]. In addition, they unvieled that substantial amounts of insulin crossed the BBB at serum levels which did not cause hypoglycemia. Thus, insulin transport and the signaling between the peripheral tissues and the brain is likely important at physiological levels of insulin as well as at higher, hypoglycemic levels. As an aside, these studies shed light on the great difficulties that the early studies faced in examining the questions of whether insulin crossed the BBB. As those studies had to elevate serum levels above the physiologic range, they were studying the transporter at its less efficient levels. The studies with radioactive insulin showed no delay in entry into brain tissue, suggesting that the delay in elevation of CSF levels may indeed be caused by the brain interstitial fluid compartment [10].

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12.2 Pathophysiology of Insulin Transport

A hallmark of BBB transport systems across the BBB is that they are not static. Instead, they are modulated by various agents and events. As such, they change during development and maturation, with the physiological demands of the CNS, and are altered in disease states. This hallmark of BBB transporters extends to those systems for peptides and regulatory proteins in general and for insulin in particular. For example, insulin transport is increased in iron-deficient rats and decreased by aluminum or dexamethasone treatment [33, 35, 36]. Insulin binding is enhanced in newborns and the brain-blood efflux of insulin is retarded by starvation and tumor necrosis factor-alpha [30, 37], whereas insulin protein and receptor levels in brain decrease with aging [38]. Most of the other effects on insulin transport can be broadly related to obesity, diabetes, insulin resistance, and Alzheimer’s disease. These are reviewed below.

12.2.1 Insulin, Obesity, and Diabetes

CNS insulin is important in brain maturation [8, 39], increases brain glucose utilization [40, 41], promotes synthesis of acetylcholine [41], alters norepinephrine and dopamine levels and turnover in selective brain regions [42, 43] in part by enhancing transport across the BBB of tryptophan and tyrosine [26], increases efferent sympathetic nerve activity [44], modulates neuronal responses in the olfactory bulb and amygdala to stimuli [45], affects pituitary sex hormone secretion [46], and alters auditory evoked potentials [47]. Many of these effects of CNS insulin are opposite to those of peripheral insulin. For example, CNS insulin induces hyperglycemia, hypoinsulinemia, and anorexia, decreases neuropeptide Y expression in the hypothalamus, and ultimately decreases body weight [48–53]. These effects are likely occurring at physiological levels of insulin, as antibodies to insulin given directly into the brain enhance feeding [51, 54] and increase bodyweight [51]. Mice with a selective knockout of insulin neuronal receptors have an increased food intake, increased serum insulin levels, insulin resistance, mild diet-induced obesity, increased serum leptin levels, hypogonadal hypogonadism, and dyslipidemia [46]. Taken together, these studies suggest that, at least for energy homeostasis, CNS insulin has a complex temporal action [55, 56]. Specifically, acute CNS insulin opposes peripheral insulin by increasing serum glucose, decreasing serum insulin, inducing insulin resistance, and reducing food intake. As CNS insulin is derivative of serum insulin, these CNS actions would be a counterbalance to the insulin released into serum in response to a meal and so would protect from hypoglycemia. Of these acute actions, decreased feeding ultimately exerts the most important chronic effect. By preventing obesity and its accompanying proinflammatory state, CNS insulin ultimately prevents the chronic insulinresistant state that can lead to type 2 diabetes [55, 56].


Insulin transport across the BBB has been found to be reduced in obese dogs and rats [57, 58]. After insulin infusions, for example, obese fafa rats had both lower CSF insulin levels and lower CSF/serum ratios than did lean fafa rats. Brain levels of insulin also decreased in the obese rats. In comparison, transport of insulin into CSF and binding of radioactive insulin to brain capillaries was not different with diet-induced obesity in the Osborne-Mendel rat [59]. However, the obese rats weighed only 20% more than the controls, a difference that may be too small to detect obesity-related differences. These findings with the BBB reinforced other work that indicated that insulin was playing an important role in energy homeostasis. Work with marmots showed that CSF insulin levels decreased early during hibernation despite maintenance of serum insulin levels [58]. This strongly suggested that insulin transport across the BBB was turned off during hibernation, a period when feeding signals were not needed. CSF insulin also decreases with fasting, strongly suggesting that insulin transport is also impaired [12]. Overall, these findings suggest that CNS insulin is important in energy homeostasis. Impaired BBB transport of insulin and the resulting decrease in CNS insulin could lead to derangements in energy homeostasis which would favor diet-induced obesity and, ultimately, insulin resistance in peripheral tissues.

12.2.2 Insulin Resistance and Inflammatory States

We have shown that treatment with lipopolysaccharide (LPS) stimulates insulin transport across the BBB [60]. LPS, also termed endotoxin, is derived from the coat of gram negative bacteria and is a powerful immune stimulant. Since CNS insulin opposes many of the actions of peripheral insulin, LPS-induced enhancement of insulin transport into the brain could underlie the mechanisms by which proinflammatory states induce insulin resistance. Indeed, CNS insulin and peripheral LPS have many similar effects. Thus, enhanced insulin transport across the BBB is one mechanism by which LPS could induce anorexia, glycemic dysregulation, insulin resistance, dyslipidemia, learning and memory deficits, impaired sexual and reproductive functions, and impairment of motivation. The actions of LPS on the BBB transport of insulin are likely mediated through the proinflammatory cytokines IL-1, IL-6, and TNF. These cytokines are known to be released by LPS and many of the effects of LPS are mediated through them. The literature supports these three cytokines in particular as playing a central role in mediating the effects of LPS on insulin transport. This is based on three lines of evidence: (1) These three cytokines have themselves been shown to mimic the effects of CNS insulin, including inducing insulin resistance, anorexia, body weight loss, dyslipidemia, cognitive effects, decreased motivation, and sexual/reproduction effects; (2) they are associated in clinical studies with insulin resistance in non-LPS-mediated proinflammatory states such as obesity, post-myocardial infarction, and lipodystrophic AIDS, and (3) they are known to alter other aspects of BBB function.

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IL-6 and TNF induce insulin resistance [61–63], cause anorexia [64, 65], decrease bodyweight [66–69], affect cognitive function and motivation [66–68, 70], and affect sexual/reproductive function [67, 68], all actions ascribed to CNS insulin. IL-1 also has effects on cognition, motivation, and anorexia and is the main mediator of sickness behavior [67, 68, 71]. Based on studies with TNF and IL-6 knockout mice, Keller et al. [62] proposed that TNF-induced insulin resistance is mediated through IL-6, suggesting a cytokine cascade. IL-6 knockout mice are protected from the development of age-related obesity, hyperglycemia, and dyslipidemia [72]. In humans, the degree of anorexia induced by LPS correlates with blood levels of IL-6 and TNF [73]. In contrast, evidence suggests that IL-1 is an important regulator of physiological insulin regulation [74] and is more associated with insulin sensitivity. IL-1 induces the release of insulin [75, 76] and mediates LPS-induced hypoglycemia in mice [77]. These studies show that insulin resistance in inflammatory states is mediated in large part through TNF and IL-6, perhaps with opposition by IL-1. Although some of these actions are likely mediated directly on various tissues, effects of insulin transport into the CNS could be another mechanism. The mentioned cytokines are associated in clinical studies with the insulin resistance in non-LPS mediated proinflammatory states such as obesity, post-myocardial infarction, and lipodystrophic AIDS. In obesity, adipose tissue is a source of TNF and interleukins [78, 79]. Insulin resistance develops in 40% of nondiabetic patients after myocardial infarction and the degree of insulin resistance correlates with serum levels of IL-6 [80]. In the Nurses Health Study of 32 826 women, serum levels of IL-6 and TNF significantly predicted the risk of developing diabetes, even after controlling for age, race, lifestyle factors, and BMI [81]. In the nondiabetic elderly, IL-1ra, IL-6 and TNF correlate with the degree of insulin resistance [82]. The finding that IL-1ra, the endogenous antagonist to IL-1, correlated with insulin resistance again suggests that IL-1 may be acting in opposition to the other two proinflammatory cytokines. Evidence suggests that the thiazolidinediones, a powerful class of antidiabetic drugs which work by enhancing insulin sensitivity, work in part by decreasing the production of proinflammtory cytokines from adipose tissue [83]. These studies show that insulin resistance occurs in proinflammatory states other than sepsis and that this insulin resistance is likely mediated by the proinflammatory cytokines. Taken together, the literature shows that IL-6 and TNF recapitulate those effects associated with CNS insulin, that insulin resistance in various proinflammatory states is affected by these cytokines, and that these cytokines have numerous effects on BBB function. IL-1 may represent the nitric oxide-mediated opposition to the LPS-simulated transport of insulin. These points make it highly likely that the ability of LPS to alter insulin transport across the BBB is mediated through one or more of these cytokines. These mechanisms likely mediate the insulin resistance of states other than just gram negative sepsis. A number of conditions associated with insulin resistance are now recognized as proinflammatory states. Obesity is the most studied example. Adipose tissue is a source of interleukins and TNF; and levels of


proinflammatory cytokines are elevated in obese persons. This proinflammatory state as mediated through the proinflammatory cytokines is currently considered the mechanism by which obesity induces insulin-resistant diabetes [55]. Moreover, a wide variety of other conditions with insulin resistance are closely linked with proinflammatory status and range from post-myocardial infarction to AIDS lipodystrophy. The American Academy of Clinical Endocrinologists and the American College of Endocrinology, based on task force recommendations, have recognized a new syndrome, referred to as the insulin resistance syndrome (IRS; in Sept/Oct 2003, Supplement 2 of volume 9 of Endocrine Practice was devoted to this topic). IRS is characterized as that compensatory phase of tissue resistance to insulin when serum levels of insulin are elevated but serum glucose levels are still in the normal range (as opposed to type 2 diabetes mellitus, in which hyperglycemia results from the inability of the compensatory hyperinsulinemia to overcome insulin resistance). IRS predisposes to or is associated with many of the same conditions [84] that form the metabolic X syndrome (which includes as one of its characteristics insulin-resistant diabetes mellitus [85]): hypertension, stroke, nonalcoholic fatty liver disease, polycystic ovary syndrome, and coronary artery disease. Furthermore, risk factors for IRS include obesity and a proinflammatory state as measured by acute phase reactants. Taken together, these findings strongly suggest that insulin resistance induced by proinflammatory conditions forms a spectrum from severe hyperglycemia to IRS. This may not be coincidental, but based on evolutionary pressures. Lazar, in a recent viewpoint in Science, pointed out that insulin resistance has survival value in stresses such as infection, possibly by making glucose less available to insulin-sensitive tissues such as muscle and therefore more available to noninsulin-sensitive tissues such as immune cells and brain [55]. This reinforces the idea that those mechanisms induced by our model of LPS administration may also be operational at lower levels of inflammation, such as obesity. Besides insulin resistance, our hypothesis also links many other conditions. This is because an increase in CNS insulin mimics many of the other findings of proinflammatory states: anorexia, cognitive effects, effects on sexual activity and reproduction, and motivation. By studying the ability of LPS to mediate transport of insulin into the CNS, one can study mechanisms that recapitulate the classic glucose intolerance of sepsis. However, these same mechanisms in attenuated form likely underlie the insulin resistance seen in other proinflammatory states including IRS and obesity.

12.2.3 Insulin and Alzheimer’s Disease

Insulin transport across the BBB may also be involved in Alzheimer’s disease (AD). Islet amyloid deposits were first described in autopsy studies [86], even before Dr Alois Alzheimer first reported the presence of “senile plaques” in 1906.

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These deposits have since been elucidated to consist of islet amyloid polypeptide (IAPP) [87], which bears a 90% structural similarity to amyloid A precursor protein [88]. This observation is particularly relevant in the light of recent epidemiological evidence, suggesting that AD may predispose to a state of insulin resistance [89], insulin hypersecretion [89], and type 2 diabetes mellitus [90]. Conversely, individuals with type 2 diabetes and hyperinsulinemia show an association with increased prevalence of AD [91, 92], as well as hippocampal and amygdalar atrophy on magnetic resonance imaging that is independent of vascular pathology [93]. A recent study also reported that diet-induced insulin resistance promoted brain b-amyloid formation in a transgenic mouse model of AD [94]. Further insight into this complex but fascinating link between AD and diabetes may be accrued from an understanding of the different mechanisms of insulin action in the CNS. On the one hand, insulin can facilitate memory via various modalities: acting in concert with glucose to increase cholinergic activity, which is greatly decreased in AD [95], facilitating long-term potentiation of memory by hippocampal synaptic plasticity, mediated by glutamate receptors [96] and insulin-sensitive glucose transporters such as GLUT4 and GLUT8 [97], and promoting neurogenesis [98]. At the same time, insulin plays a pivotal role as a neuromodulator involved in the regulation of vital homeostatic processes essential for cerebral function. Insulin dysregulation may therefore contribute to AD pathology through several mechanisms, including: oxidative stress through the formation of advanced glycation end-products [99], increased tau phosphorylation and neurofibrillary tangle formation [100], and increased b-amyloid aggregation through competitive inhibition of insulin-degrading enzyme (IDE), a metalloprotease present on brain microvessels that degrades both insulin and b-amyloid [101]. This field has considered whether the effects of insulin on the brain should be regarded as mainly an extension of its peripheral action (in other words, as a regulator of systemic energy homeostasis), or whether insulin exerts CNS effects that are clearly independent of its peripheral metabolic effects. Currently, the weight of evidence favors the latter viewpoint. Thus, findings in this field resonate with current viewpoints in the area of energy homeostasis. Consistent with the notion of regional variation in insulin receptor density within the CNS [102], studies have indicated the prevalence of insulin receptors in the limbic and hippocampal regions [103]. A further extension of the concept of regional specificity is seen in the selective function of insulin receptors in these regions, where they have been shown to be involved in synaptic plasticity and long-term memory consolidation [104]. Accruing evidence also corroborates the contrasting effects of acute compared with chronic elevation of glucose and insulin in the brain [95]. Acutely raising plasma insulin levels, while maintaining euglycemia, can improve memory in Alzheimer’s patients [105]. However, raising glucose levels while suppressing endogenous insulin secretion abolished the memory enhancing effects of glucose, suggesting the pivotal role of insulin-mediated memory facilitation in AD [106]. At first sight, this may seem surprising, as both chronic hyperinsulinemia and


chronic hyperglycemia are associated with accelerated cognitive decline in the elderly [107]. However, this apparent discrepancy should be seen in the light of the mechanisms underlying spontaneous chronic hyperinsulinemia and chronic hyperglycemia, of which insulin resistance is an important denominator. Consistent with these roles are two studies which have examined the ability of insulin to cross the BBB in animal models of Alzheimer’s disease. One model used the SAMP8 mouse, a natural mutation which has an age-dependent overexpression of amyloid precursor peptide, age-dependent increases in amyloid beta protein, and age-dependent learning and memory deficits which are reversed by antibodies or anti-sense directed against amyloid beta protein [108, 109]. Uptake was higher in aged SAMP8 mice for the cerebellum and the thalamus [110]. A second model used double-transgenic mice which overexpressed both amyloid precursor protein and presenilin [111]. This model found higher uptake of insulin in four of six brain regions. This brings us to the last related point of the influence of disease-dependent factors on CNS insulin action. Converging evidence suggests that impairment at the level of the BBB in the insulin signal transduction cascade, and possibly insulin growth factor-1 signaling, may be an early and pivotal event in the pathogenesis of AD [112]. In this instance, although neuronal insulin receptors are upregulated [113], they exhibit defective signal transduction, analogous to the peripheral insulin resistant state seen in type 2 diabetes mellitus. This has led to the hypothesis that AD may be the “insulin-resistant brain state” equivalent of noninsulin-dependent diabetes mellitus [114]. This is an attractive hypothesis, which may also account for the apparent differential effects of acute and chronic hyperinsulinemia in AD [115]. Acute administration of exogenous insulin may serve to temporarily overcome cerebral insulin resistance, thereby improving memory. In contrast, chronic “endogenous” hyperinsulinemia reflects a compensatory response to insulin resistance of peripheral tissues. The seemingly elevated insulin levels belie a state of relative insulin deficiency that prevails when this compensatory response becomes inadequate. In support of this hypothesis is the observation that peripheral insulin infusion in humans increases the levels of b-amyloid in cerebrospinal fluid, indirectly supporting the putative role of hyperinsulinemia in the pathogenesis of AD [116]. In addition, different mechanisms appear to be involved within the nosology of AD [117]. In early onset familial AD (ca. 5% of all AD cases), defective signal transduction is due to an insulin-binding deficit at the a-subunit of the insulin receptor that results from the competitive binding of b-amyloid [118]. In late-onset sporadic AD (95% or more of cases), though, the age-related increase in CNS noradrenaline and cortisol levels inhibits the b-subunit of the neuronal insulin receptor at different sites, leading to receptor desensitization [117]. There is also evidence that the apolipoprotein E (APOE) genotype, a prominent genetic factor in sporadic AD, can modulate the risk for insulin resistance [119] and affect insulin degrading enzyme levels [120]. Novel strategies based on this common pathologic link between insulin and cerebrovascular Ab amyloidoses are being explored for the treatment of AD.

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These include the enhancement of Ab-degrading enzymes, such as insulindegrading enzyme [121], and insulin sensitizers such as rosiglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist [122]. Although intranasal insulin has been shown to improve short-term memory and mood in healthy subjects without systemic effects [123], it is uncertain whether chronic administration in AD patients would aggravate cerebral insulin resistance and thereby impede cognition in the long term.

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Anon. 2003, Am. J. Physiol. 162, 313–319. Turner, A. J., Fisk, L., Nalivaeva, N. N. 2004, Ann. N.Y. Acad. Sci. 1035, 1–20. Watson, G. S., Craft, S. 2004, CNS Drugs 17, 27–45. Benedict, C., Hallschmid, M., Hatke, A., Schultes, B., Fehm, H. L., Born, J., Kern, W. 2004, Psychoneuroendocrinology 29, 1326–1334.

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13 Glucocorticoid Hormones and Estrogens: Their Interaction with the Endothelial Cells of the Blood-Brain Barrier Jean-Bernard Dietrich

13.1 Introduction

The blood-brain barrier (BBB) isolates the central nervous system from the blood. The BBB is mainly composed of capillary endothelial cells, which normally block nonadherent circulating leukocytes in the bloodstream, but permit their transmigration into neighboring tissues following an inflammatory stimulus. Many adhesion molecules are known to be necessary for this recruitment of leukocytes. The expression of these adhesion molecules in brain-derived microvascular endothelial cells can be regulated by glucocorticoid hormones. This important class of hormones has proven particularly useful in the treatment of multiple sclerosis, a disease resulting from the chronic inflammation of the central nervous system and mediated by the infiltration of inflammatory cells of the immune system. Estrogens can also modulate the expression of these endothelial adhesion molecules, thus regulating the leukocyte-endothelial cell interactions. In addition, estrogens are useful for the treatment of experimental autoimmune encephalomyelitis, an animal model with similarities to multiple sclerosis (MS). In this chapter, the effects of glucocorticoids (GC) and estrogens on brain endothelial cells and their use as therapeutic tools for the treatment of autoimmune diseases are discussed. There is no doubt that GC hormones probably affect every organ in mammals, but many of their effects are specific for particular cells or tissues. GC mediate their action through a cytoplasmic receptor which has several functional domains and activation of this receptor directly or indirectly regulates the transcription of target genes [1, 2]. The brain is a major target tissue for GC and these hormones interact with endothelial cells, especially those of the BBB. These BBB endothelial cells can be distinguished from others in the body by the absence of fenestrations and more extensive tight junctions, which actually limit the flux of hydrophilic molecules across the BBB [3, 4]. Blood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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13 Glucocorticoid Hormones and Estrogens

Dysfunction of the BBB is a pathologic event associated with several diseases, including MS, Alzheimer’s disease and HIV-associated dementia. An alteration in the integrity of the BBB allows the migration of activated leukocytes into the brain. GC are widely used to treat MS and autoimmune diseases in general, because they have potent anti-inflammatory and immunosuppressive properties, and beneficial effects on the maintenance of the BBB. Estrogens are also involved in the regulation of genes of these brain endothelial cells and modulate several of their biological activities, including their interaction with leukocytes. In the normal brain, endothelial cells lining the microvasculature limit the entry of leukocytes. Estrogens act via estrogen hormone receptors (ER), which are transcription factors regulating the expression of their target genes [5, 6]. These hormones, like GC, are involved in the regulation of the expression of adhesion molecules in brain microvascular endothelial cells and consequently regulate their interaction with activated leukocytes. The effects of GC and estrogens on endothelial cells of the BBB are reviewed and their use in the therapy of some neurodegenerative diseases is discussed.

13.2 Glucocorticoids and the Endothelial Cells of the BBB 13.2.1 Mechanisms of Action of Glucocorticoids

As there are now many detailed and updated reviews dealing with GC mechanisms of action [7, 8], only a short survey is presented here. One can actually distinguish two modes of action of GC: genomic and nongenomic. It is well known that GC passively diffuse into the cell and can bind to the intracellular glucocorticoid receptor (GR). GR is maintained in an inactive conformation by a heat-shock protein (HSP) complex including hsp 90, hsp 70, and immunophilin FKBP59. The interaction between the steroid hormone ligand and the GR activates the receptor and the HSP complex is dissociated from the GR. Consequently, a homodimer of two activated GRs can be formed, which is then transported to the nucleus. This homodimer will bind to specific GC-responsive elements (GRE) and regulate the expression of target genes. The GC-GR complex induces direct gene transactivation and influences both translational and posttranslational processes [9, 10]. In addition, considerable cross-talk occurs between GR and other transcription factors (NF-kappaB, AP-1) by direct protein-protein interactions [11]. Transcription of the genes coding E-selectin, ICAM-1, VCAM-1, and PECAM-1 has been shown to be regulated by NF-kappaB, a transcription factor induced by pro-inflammatory cytokines [12–14]. Repression of NF-kappaB signaling by GC is the mode of action by which these hormones counteract the expression of cellular adhesion molecule (CAM) genes induced by various factors such as cyto-

13.2 Glucocorticoids and the Endothelial Cells of the BBB

kines [15]. Thus GC negatively influence pro-inflammatory transcription factors. NF-kappaB can be repressed by GR, either by directly reducing its activity, or indirectly by induction of I-kappaB, an inhibitor of NF-kappaB [16]. These transrepression mechanisms mediate the anti-inflammatory effects of GC and provide a molecular basis to their therapeutic properties. Nongenomic pathways are another aspect of GC action. Some very rapid effects of GC cannot be explained by the classic genomic theory of GC action and it is possible that nongenomic actions are mediated by a distinct membrane receptor [17]. Two types of nongenomic effects are documented: specific and nonspecific. Nonspecific effects occur within seconds only at high GC dosage and seem to result from direct interactions with biological membranes. Specific effects are observed within a few minutes and are mediated by steroid-specific membrane receptors. These receptors possess distinctive hormone-binding properties, compared with the well known cytoplasmic receptors, and are associated with the caveolae components of the plasma membrane. Interestingly, a new receptor conformational ensemble model describes how a classic GC receptor could accommodate differently shaped ligands to result in the initiation of either rapid or genomic responses [17]. An interesting example of a nongenomic pathway is the effect of GC on the stimulation of ecto-apyrase (CD39) activity observed in stimulated endothelial cells or mesangial cells in vitro. In this study, prednisolone was able to reduce CD39 expression, an effect which may paradoxically lead to reduced hydrolysis of pro-inflammatory or pro-aggregatory mediators (like extracellular ATP or ADP, respectively). The downregulation of this enzyme, which belongs to the family of ectonucleoside phosphatases, seems to promote the presence of these pro-inflammatory nucleotides. An identical effect was observed when dexamethasone (DEX) was used instead of prednisolone. Mifepristone (RU 486), which is an antagonist of cytoplasmic GR, did not block the effect of prednisolone, suggesting that its effect on soluble apyrase is at least partly mediated through a nongenomic pathway [18]. Recently, it was shown that DEX is able to induce rapid serine phosphorylation and membrane translocation of annexin-1 (ANXA-1) via a novel nongenomic, GC-dependent mechanism, requiring MAP kinase, phosphatidylinositol 3-kinase, and Ca2+-dependent PKC pathways [19].

13.2.2 Glucocorticoids and Inflammation

Inflammation is a response of the body to tissue irritation or injury. The work of E. Metchnikoff at the end of the 19th century provided a description of the basic aspects of inflammation [20]. The present understanding of the inflammatory response highlights the molecular mechanism involved in the activation and recruitment of lymphocytes from the circulation and their trafficking through the body. Inflammatory mediators have been identified and the importance of adhesion molecules in the control of acute inflammation has been

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demonstrated. Acute inflammation is a phenomenon where rapid infiltration of polymorphonuclear neutrophils is followed by monocytes and eventually lymphocytes [21]. Leukocyte recruitment into extravascular tissue is indeed regulated by specific combinations of adhesion and signaling molecules [22–24]. GC are able to exert their anti-inflammatory effect by inhibiting the expression of the genes coding cytokines and adhesion molecules as well as by redirecting the traffic of lymphocytes [25, 26]. Expression of inducible nitric oxide synthase, an enzyme required for nitric oxide (NO) synthesis, is repressed by GC [27]. NO synthase is known to contribute to the inflammation process [28]. GC also have a direct effect on the expression of lipid mediators of inflammation, because they can inhibit the activity of enzymes involved in their biosynthesis. A good example is the regulation of cytokine-induced cyclo-oxygenase 2 by DEX [29].

13.2.3 Regulation of Adhesion Molecules Expression by GC in Endothelial Cells

The adhesion molecules involved in leukocyte recruitment are well documented: they include selectins, integrins, and members of the immunoglobulin superfamily (ICAM-1,-2, VCAM-1, PECAM-1) and CD 99 (Table 13.1). Leukocytes emigrate from the bloodstream into inflamed tissues by sequential events. These intercellular adhesion events with endothelial cells lining the vascular wall are now well characterized and described in many papers [24, 30–32]. A general paradigm for this process is now accepted. The first steps, tethering and rolling, are mediated by the members of the selectin family: L-selectins expressed mainly by the leukocytes, and P- and E-selectins expressed by platelets and endothelial cells respectively. The counter-ligands of the selectins are sialylated, fucosylated receptors. However, some leukocytes use the interaction between alpha 4-beta 1 integrin (VL4, CD49d-CD29) and vascular cell adhesion molecule 1 (VCAM-1) for both these steps. The second action, rolling, brings the leukocyte into close contact with the endothelium, which can then be activated by chemokines [33], platelet activating factor [24], or other soluble or membrane-bound molecules. These signals activate the beta 2 integrins (CD11– CD18 family) specific for leukocytes and allow the firm adhesion of these cells on the endothelial luminal surface. The counter-receptors on endothelial cells are members of the immunoglobulin gene superfamily. Intercellular adhesion molecules (ICAM-1, ICAM-2) are involved as well as VCAM-1. Leukocytes then squeeze between the opposing endothelial cells and this last step is known as transmigration or diapedesis. Homophilic interactions between the same molecules expressed on both endothelial cells and leukocytes mediate this process. PECAM-1 (CD31) plays an important role in the transmigration of monocytes, neutrophils, and natural killer cells, both in vitro [34, 35] and in vivo [36]. Diapedesis and subsequent migration across the basal lamina and interstitial tissues are stimulated by soluble and membrane-bound chemoattractants from the inflammatory environment (bacterial cell products, chemokines synthesized by


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Table 13.1 Adhesion molecules involved in inflammation: leukocyte adhesion molecules and their ligands on endothelial cells, with CD and integrin nomenclatures in brackets. E-Selectin ligand-1, CLA bearing sialylLewisx P-selectin E-selectin (CD62P) (CD62P)

VLA-4 (CD49d/ C D18; a4b1)

LFA-1 (CD11a/ C D18; aLbL2)

VCAM-1 (CD106)

T, R

T, R tight adhesion (TA) N, M, B&T, NK

Leukocyte adhesion molecules

L-Selectin (CD62L)

PSGL-1 (CD162)

Endothelial cell counter-ligand

Sialyl-Lewisx on appropriate ligand

Actions

Tethering (T), rolling (R)

Leukocytes involved

N, M, Neutrophils (N), N, M, monocytes (M), B&T, NK B&T, NK lymphocytes B&T (B&T), natural killer cells (NK)

T, R

PECA M-1 (CD31)

(CD99)

ICAM-1 (CD54), ICAM-2 (CD102) TA

Mac-1, CR3 (CD11b/ CD18; aMb2) ICAM-1 (CD54)

PECA M-1 (CD31)

(CD99)

TA

Diapedesis (D)

D

M, B&T, NK

N, M, NK

N, M, NK, subsets of T

N, M, B&T, NK

host cells in response to inflammatory stimuli). The role of a chemoattractant gradient was recently documented [37]. CD99 also plays a major role in the migration of monocytes through endothelial junctions [38]. In addition, the molecule JAM-1 (junctional adhesion molecule 1) has been identified and shown to be involved in tight adhesion or transmigration of leukocytes, depending on its apical or junctional localization on endothelial cells. JAM-1 is involved in such interactions via JAM-1/LFA-1 contacts [39, 40]. How do GC act on the expression of these adhesion molecules at the level of endothelial cells, especially the endothelial cells of the BBB? This is an important question, not only on a fundamental level but also in relation to the anti-inflammatory properties of GC. Among GC, DEX is a synthetic glucocorticoid used as a potent anti-inflammatory product and also for studies of gene expression in many cell lines or tissues. The effects of DEX and another GC, methylprednisolone (MP), on the expression of the genes coding these adhesion molecules are summarized in Table 13.2. Expression of E-selectin is upregulated by cytokines and lipopolysaccharides (LPS) in human brain microvessel endothelial cells (HBECs) [15] and by TNF-alpha and LPS in aortic endothelial cells and in HUVECs. This effect is markedly reduced in the presence of DEX, both in aortic endothelial cells [41] and in HUVECs [14]. Inflammatory cytokines augment synthesis of P-selectin in human tissues affected by allergic or chronic inflammation [42, 43]. In human endothelial cells, the interleukin-4-induced expression of P-selectin, which is constitutively synthesized by endothelial cells, is not affected by DEX but is decreased by the proteasome inhibitor N-acetyl-leucinyl-leucinyl-norleucinal-H, the antioxidant pyrrolidine dithiocarbamate, or sodium salicylate [44]. DEX can also induce

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Table 13.2 Adhesion molecules expressed in endothelial cells (ECV304: human endothelial-like cells; EA hy926: human umbilical vein cell line; HBECs: human brain endothelial cells; HUVECs: human umbilical vein endothelial cells) and involved in leukocyte recruitment at the BBB. Effects of GC. Name of cell or cell line

Name of gene

Description

Inducers of gene expression

GC used (effects on gene expression)

Ref.

HBECs, HUVECs

E-selectin, ELAM-1

TNF-alpha, interleukin-1beta, LPS

DEX (downregulation)

14, 15, 41

HUVECs HUVECs, EAhy926 ECV304

P-selectin ICAM-1 (CD54)

Selectin: endothelial leukocyte adhesion molecule Selectin Intercellular adhesion molecule Intercellular adhesion molecule

Interleukin-4 LPS

DEX (no effect) DEX (downregulation) DEX (downregulation)

44 46–48

HBECs, HUVECs

ICAM-1 (CD54)

Intercellular adhesion molecule

51, 52

HBECs

VCAM-1 (CD106)

Vascular cell adhesion molecule

DEX, MP (high doses; downregulation) MP (high doses; downregulation)

ICAM-1 (CD54)

Thyroid hormone, T3(3,5,3'triiodothyronine) TNF-alpha

TNF-alpha

50

51, 52

the shedding of L-selectin in the monocytic cell line U-937, which may contribute to the anti-inflammatory action of GC [45]. ICAM-1 expression, which can be stimulated by LPS, is inhibited by DEX in HUVECs and in the human vascular endothelial cell line EAhy926, a hybridoma of HUVECs and the human epithelial cell line A549 [46–48]. The same effect was found in the ECV304 cells, a human endothelial-like cell line [49] where ICAM-1 expression stimulated by the thyroid hormone T3 or interleukin1beta or LPS was significantly inhibited by DEX [50]. The effects of methylprednisolone (MP) on brain endothelium has been studied at low (65 lg ml–1) and high (650 lg ml–1) doses and compared with the effects of DEX. In HBECs and HUVECs, the effects of GC were observed both in basal conditions and after stimulation with TNF-alpha or interferon-gamma. In this work, only high-dose MP treatment downregulated TNF-alpha-induced VCAM-1 expression. Neither TNF-alpha-induced ICAM-1 expression nor interferon-gamma-induced HLA-DR expression in either cell lineage were influenced by DEX or MP treatment [51]. However, the same research group showed that TNF-alpha-induced ICAM-1 and VCAM-1 expression in HBECs was reduced by treatment with high doses of MP [52]. Intravenous administration of MP also altered the kinetics and distribution of monocyte adhesion molecule expression as well as endothelial cell release of E-selectin and this may limit monocyte recruitment to areas of tissue destruction in MS [53].


13.2.4 Effects of GC on Leukocyte-Endothelial Cell Interactions

Cell adhesion molecules provide the foundation for cell-cell interactions, trafficking, and immune surveillance, which is essential to host defense. Leukocyteendothelial cell interactions initiate the recruitment of leukocytes from the bloodstream to inflamed tissues. A prominent feature of many neurodegenerative diseases is the dysfunction of leukocyte transmigration, for example in MS. For this reason, understanding normal leukocyte-endothelium interactions is an important step for the development of potential therapies of inflammation. Animal models serve as an alternative approach to develop strategies to overcome tissue damage induced by excessive leukocyte infiltration [54]. Model systems allow leukocyte trafficking across the BBB to be studied in vitro [55, 56]. From the information obtained from these models, opportunities for clinical translation have emerged, for example the use of monoclonal antibodies directed against specific adhesion molecules, like integrins or VCAM-1, for the treatment of these diseases [57]. The effects of GC on T cell recruitment across the BBB were examined recently [58]. High-dose methylprednisolone (MP) treatment was found to significantly reduce soluble ICAM-1 level, but not soluble ICAM-3 after 10 days in MS [59, 60]. In addition, the essential role of ICAM-1 and ICAM-2 was demonstrated in transendothelial migration of T cells using an in vitro model composed of endothelioma and mouse T cells [61]. In particular, ICAM-1 and ICAM-2 were found to be unique due to their interaction with the cytoskeleton. ICAM-1 and ICAM-2 have been shown to bind directly to alpha-actinin [62] and ezrin, a member of the ezrin-radixin-moesin (ERM) family, was found to act as a linker between cell surface components and the cytoskeleton by direct binding to the cytoplasmic tail of ICAM-1 and ICAM-2 [63]. Ezrin can influence ICAM-2 surface distribution [64]. GC inhibition of cellular adhesion molecule expression seems to lead to a reduced adhesion of the leukocytes to the endothelium. The relevance of this hypothesis to humans has been confirmed, because GC treatment was found to decrease infiltration of peripheral blood mononuclear (PBMN) cells at sites of inflammation with a concomitant peripheral leukocytosis [65] and high-dose MP treatment reduced adhesion of leukocytes to endothelium [66]. A detailed analysis of the effects of GC on leukocyte-endothelial cell interactions clearly indicates the complexity and the multifactorial aspects of the mechanisms modulating this regulation via GC [67]. These authors concluded that GC can act on target cells at multiple levels. Regulation of CAM by GC depends both on the type of cell and the stimuli considered. Both genomic and nongenomic mechanisms are operative [68]. In addition, GC can act by either inducing anti-inflammatory molecules or inhibiting pro-inflammatory mediators (cytokines, chemokines). With respect to the synthesis of anti-inflammatory mediators, annexin-1 (ANXA-1, previously referred as lipocortin-1) was investigated and has been proposed to act as a putative mediator of anti-inflammatory effects of GC. This 37-kDa protein found in the cytosol of many cell types is up-

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regulated by GC on circulating leukocytes [69]. ANXA-1 is involved in the regulation of all steps of the adhesion cascade [67, 70]. ANXA-1 does not affect CAM gene expression on either resting or activated lymphocytes [71]: this protein influences leukocyte adhesion events by modulating CAM functions but not by regulating CAM gene transactivation. ANXA-1 in vivo effects on leukocyte adhesion and transmigration cause conformational/functional changes of surface CAM. Thus, these effects can be considered as nongenomic [72]. Finally, induction of CAM gene expression may be regulated in different cell types by various cytokines and chemokines. This highlights the inhibitory effect of GC on pro-inflammatory mediators. For example, GC have been shown to inhibit lymphocyte CAM by interfering with a set of IL-2-gamma-dependent cytokines [73].

13.2.5 Glucocorticoids, Cerebral Endothelium and Multiple Sclerosis

GC effects on brain vascular endothelial cells are not only observed at the level of the regulation of adhesion molecules expression. Other genes are under GC control. In human brain-derived microvascular endothelial cells (HBECs), DEX (1 lM) treatment for 24 h decreased the number of high affinity endothelin-1 binding sites. In this way, GC may counteract some endothelin-induced events in the cerebral endothelium (permeability changes, adhesion molecule expression) which are involved in the development of inflammatory and/or cerebrovascular brain disorders [74]. Statins induce apoptosis in many cell types. The effect of fluvastatin (FS) at a micromolar concentration was tested on the endothelial cell line EA.hy926. DEX (1 lM) blocked FS-induced apoptosis as well as apoptosis induced by serum deprivation, TNF-alpha, oxidation, DNA damage, and mitochondria disruption. This study suggests that GC play a role in preventing vascular injury and explains why statins are not toxic to vascular endothelial cells in vivo [75]. Histamine H1 and H2 receptors in rat brain endothelial cells (RBE4 cells) are downregulated by DEX at the mRNA level. This mechanism may be involved in GC-mediated effects on cerebrovascular permeability and brain edema [76]. DEX decreases transmonolayer permeability in cultured rat brain endothelial cells: this can be demonstrated using sucrose, fluorescein, and dextrans of up to 20 kDa in GPNT rat brain endothelial cells treated with 1 lM DEX. The continuity of the tight junctional protein zonulae occludens-1 (ZO-1) is characteristic of the BBB at inter-endothelial cell-cell contacts. A more regular and continuous cortical ZO-1 distribution was observed following DEX treatment. Thus, these results suggest that GC can induce a more differentiated BBB phenotype in these cells by affecting tight junction (TJ) structure [77]. HBECs under basal conditions produce interleukin-8 (IL-8) and monocyte-chemoattractant protein-1 (MCP-1), as shown by the expression of their mRNA transcripts [78]. Beta-chemokines play an important role in the recruitment of mononuclear cells into the brain during a variety of neuroinflammatory conditions,


such as experimental allergic encephalomyelitis (EAE) and MS [79, 80]. Monocyte migration could be significantly inhibited by the addition of blocking antibodies to MCP-1 using HBECs in an in vitro model of the migration of cells of systemic immune origin across the BBB during the initiation of an inflammatory response within the central nervous system [81]. High-affinity saturable binding sites for MCP-1 are present along human brain microvessels [82]. This suggested that the CC chemokine receptor (CCR2) is expressed by the brain endothelium [83]. Both occludin and ZO-1 are part of TJs and disruption of the TJs could foster leukocyte extravasation. In addition to its chemotactic activity, MCP-1 has been shown to alter BBB integrity during CNS inflammation. Exposure to MCP-1 caused a loss in ZO-1 immunoreactivity at the inter-endothelial junctional region in both isolated brain microvessels and cultured brain microvessel endothelial cells (BMECs). A similar effect on occludin was observed in cultured BMECs. Finally, expression of caveolin-1, a major structural component of membrane microdomains thought to be functionally complexed with TJs, was also altered when MCP-1 was added to BMECs and microvessels [84]. Considering the pivotal role of MCP-1 in the transendothelial migration of lymphocytes, it is worth noting that DEX treatment reduces the upregulation of secreted MCP-1 after activation with TNF-alpha, IL1-beta, and IFN-gamma. This effect of DEX has been observed in vitro, using rat brain (GP8/3.9) and a retinal (JG/2) vascular endothelial cell line expressing MCP-1. In this study, DEX treatment (1 lM) significantly inhibited MCP-1 secretion in GP8/3.9 endothelial cells stimulated by TNF-alpha, IL1-beta, and IFN-gamma, by 22.7±2.4%, 24.5±6.4%, and 31.2±4.7% respectively. These in vitro findings support the hypothesis that CNS endothelial cells play an active role in the recruitment of inflammatory cells from the circulation. This could be mediated by enhanced expression of chemokines like MCP-1. Downregulation of endothelial derived MCP-1 by DEX found in vitro may account for the beneficial effects of GC observed in vivo in the treatment of autoimmune neuroinflammatory diseases [85]. Matrix metalloproteases are also present in the endothelial cells of the CNS, where their altered expression may contribute to the disruption of the BBB. DEX can partially inhibit MMP-9 cytokine-upregulated expression in rat brain cell lines like GP8/3.9. The effect of GC on MMP production may be one of the possible mechanisms by which steroids affect BBB permeability [86]. MMP production is elevated in MS patients and has a role in BBB disruption in MS. In this case, GC also inhibit the activity of these MMP [87]. GC are currently used as immunosuppressive and anti-inflammatory drugs (with quite variable doses ranging from very low to extremely high, depending on the disease). GC pulse therapy, which allows a strong induction of leukocyte apoptosis, is now the standard approach used for the treatment of neuroinflammatory diseases [88]. Apoptosis in this case contributes to the downregulation of T cell activity and terminates the inflammatory response. MP augments apoptosis of T cells in situ and a clear dose-response effect is observed in treatment of adaptative transfer EAE [89]. In vivo, unstimulated peripheral leukocytes shows enhanced apoptosis in the three groups of MS patients [90].

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The use of interferon, particularly IFN-beta, for the treatment of MS is now well documented [91–94]. The effects of GC and type I interferons (IFN-alpha, IFN-beta) used simultaneously were investigated in vivo. Both drugs additively decreased BBB permeability, but did not prevent the increase of permeability induced by a pro-inflammatory stimulus (LPS). Thus, the beneficial effect of this treatment for MS did not seem to be mediated through a direct action at the level of BBB. A more general sensitivity is involved, because pretreatment by type I IFNs potentiated the effect of GC by two orders of magnitude. Type I IFNs may possibly restore the dysfunctional T helper I (Th1/Th2) balance associated with MS [95]. IFN beta has been approved for the treatment of MS and has been demonstrated to stabilize BBB integrity in vivo. Using a coculture model (brain endothelial cells with astrocytes), Kraus and coworkers showed that recombinant IFN-beta inhibited an increased paracellular permeability for small tracers. Permeability changes were accompanied by minor changes in the staining of TJassociated proteins (occludin, claudin-3, -5, ZO-1, -2) in brain endothelial cell monolayers. Under conditions with low paracellular permeabilities, the monolayer resembled a typical cobblestone-like pattern and the TJ-associated proteins at the cellular borders appeared continuous and homogeneous. In culture conditions leading to increased permeabilities, the staining appeared to be dispersed or concentrated at the cell-cell contact zones of endothelial cells [94]. High-dose GC therapy is currently used in MS relapses and one mode of action of GC could be their inhibition of the cytokine-induced expression of CAM, which mediates leukocyte-BBB interactions and finally chronic recruitment of leukocytes across the BBB. The severe side-effects of long-term GC therapies are well known [95, 96]. To avoid these side-effects, the development of new steroidal drugs has been undertaken with the hope that these drugs could be beneficial for MS patients. These molecules would specifically transrepress NFkappaB-mediated expression of CAM [58, 97]. For cell-specific drug delivery, endothelial cells were considered as attractive targets, especially due to their direct contact with the bloodstream. To deliver GC into activated endothelial cells at the inflammatory site, DEX was conjugated to a monoclonal antibody recognizing E-selectin. It was demonstrated that this immunoconjugate was internalized by activated, not resting, endothelium via the lysosomal pathway. After intracellular degradation, pharmacologically active DEX was released, which was able to downregulate IL-8 gene [98]. In a recent study using HUVECs, the conjugate (10 lg ml–1) was found to decrease the levels of ICAM-1 and VCAM-1 after 24 h stimulation with TNF-alpha. However, the expression of 24 genes was downregulated (by 50% or more) by free DEX and the immunoconjugate after 6 h stimulation with TNF-alpha, showing that intracellularly delivered DEX is able to exert its pleiotropic anti-inflammatory effect [99].

13.3 Estrogens and the Endothelial Cells of the BBB

13.3 Estrogens and the Endothelial Cells of the BBB 13.3.1 Mechanisms of Action of Estrogens

Many recent reviews dealing with the mechanisms of action of estrogens (ES) are available [100–103]. Because the cerebral vasculature is an important target tissue for these hormones, a precise understanding of their effects at the molecular level is required. ES can act by either direct or indirect genomic pathways as well as by nongenomic effects, allowing multiple actions in a single cell. The direct genomic mechanism involves two nuclear estrogen receptors (ER): ER-alpha and ER-beta. The formation of the ES-ER complex and its dimerization leads to activation and permits association with an estrogen response element (ERE) or with an ER-dependent AP1 response element. In the latter case, the complex binds to fos-jun heterodimers which then bind to the AP-1 response element [104]. This complex thus acts as a transcription factor at the genomic level. Indirect genomic effects include activation of ER-linked second-messenger systems such as adenylyl cyclase, protein kinases A and C and MAP kinase (AC/PKC, cAMP/ PKA, MAPK/ERK). Consequently, many cell substrates are phosphorylated. Among them are transcriptional regulators like CREB which in turn interact with the DNA regulatory CRE (cAMP-responsive element). This can then allow indirect regulation of the expression of genes (without ERE). Nongenomic effects can occur at low or high estrogen concentrations, either at nanomolar or lower levels or at micromolar concentrations. These nongenomic effects are characterized by rapid changes in the cell physiology that occur on the order of minutes. At high concentrations, nongenomic effects involve antioxidant effects not mediated by known ERs [105]. The question remains whether these changes are mediated by a modified form of nuclear ER that is associated with the plasma membrane (mER) or a completely novel membrane receptor. Recently, a membrane-associated receptor was characterized in a rat hypothalamic endothelial cell line (D12 cells). These brain-derived endothelial cells express a membrane-associated protein (67 kDa molecular mass, similar to that of ER-alpha), colocalized with caveolae-enriched membranes. However, the data suggest that this mER, related to nuclear ER-alpha in D12 cells, is nevertheless biochemically and immunologically distinct [106]. At the lower estrogen concentrations, nongenomic effects have been described in neurons, and at high estrogen concentrations, neuroprotective effects have been reported for a number of cell culture models, but not endothelial cells [105]. A good example of nongenomic action of ES is vasodilation, induced in a matter of seconds to minutes. This rapid effect is a result of the release of vasoactive molecules which regulate ionic fluxes on smooth muscle and vascular endothelial cells [103, 107].

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13.3.2 Endothelial Cells as Targets of Estrogens

Indeed, the variety of ES effects on the cell, and the fact that ES can be ferried through the bloodstream to specific addresses, highlight endothelial cells as an important target for these hormones. Several ES actions are now described on cerebral vessels or endothelial cells. 17beta-estradiol (E2) is known to be a neuroprotective hormone as it protects cultured neurons against a wide range of insults. This effect is also observed in vivo [108]. For example, E2 and a low concentration of tamoxifen promoted cytoprotection of cultured rat cerebral endothelial cells treated with 3-nitropropionic acid, a mycotoxin inducing brain damage [109]. Maintenance of endothelial nitric oxide synthase (eNOS) activity is critical to vascular function, as diminished NO availability contributes to endothelial dysfunction [110]. Along these lines, it is of interest to note that chronic treatment with E2 increased eNOS mRNA and protein levels in vivo [111]. This activation may occur via the phosphoinositolkinase-Akt pathway in human endothelial cells [112, 113]. In addition, E2 modulates glucose transporter-1 protein and mRNA in the BBB endothelium. E2 treatment causes a dose- and time-dependent increase in GLUT-1 protein expression in microvessels, thus facilitating the transport of glucose, the primary fuel of the mammalian brain, across the BBB [114]. Interestingly, E2 inhibits IFN-gamma-induced class II major histocompatibility complex (MHC) expression by a novel mechanism, involving the modification of the histone acetylation status of the class II MHC promoter. One has to keep in mind that aberrant expression of class II MHC is suspected to be one of the factors leading to some autoimmune diseases, such as multiple sclerosis [115]. The presence of ER-alpha has been detected in female rat cerebral vessels and the localization of this receptor in endothelial cells allowed the detection of multiple forms of ER-alpha. Chronic exposure to E2 significantly increased the expression of these forms at the protein level [116]. In addition, HUVECs and human coronary artery cells also contain low levels of ER; and E2 also upregulates the expression of these ER [117]. Recently, using differential display analysis, three other genes were found to be upregulated by E2 in female aortic endothelial cells. Significant increases in mRNA expression (more than 2-fold) were measured for aldose reductase, caspase homologue-alpha protein, and plasminogen activator inhibitor-1 intron e. These genes may be of potential importance for vascular function in human endothelial cells [118]. These few examples demonstrate the importance of endothelial cells as targets for ES action.

13.3.3 Adhesion Molecules are Regulated by Estrogens in Endothelial Cells

One of the most important events in inflammation is clearly the adhesion of leukocytes to the endothelium, followed by their emigration through the BBB. These events are regulated by the precise interactions of complementary adhe-

13.3 Estrogens and the Endothelial Cells of the BBB

sion molecules on leukocytes and endothelial cells [23, 30]. For these reasons, understanding the effects of ES on the expression of these adhesion molecules is crucial, to determine whether ES affects the immune response in a suppressive or facilitative way. There are conflicting reports dealing with the effects of ES on the cytokine-induced expression of adhesion molecules in endothelial cells. For example, E-selectin expression is enhanced by E2 at a physiological concentration (10 nM) during the first hours of exposure to TNF-alpha in TNFalpha-stimulated HUVECs. VCAM-1 expression is also enhanced by E2 at 24 h [119]. However, after longer exposure periods (48 h) and at pharmacological doses, ES decreases the expression of cytokine-induced CAM genes. E2 strongly inhibits (60–80%) E-selectin, ICAM-1, and VCAM-1 induction in HUVECs activated by interleukin-1 [120]. E2 downregulates the E-selectin promoter through either ER-alpha or ER-beta, requiring the NF-kappaB site at positions –94 to –85 within the promoter [121]. These two examples clearly show that the dual effects of ES depend on the time and dose of exposure. E2 is also able to increase the expression of beta 1, alpha 5 and alpha 6 integrin mRNA in HUVECs, an increase followed by a later enhancement in the surface expression of these integrins [122]. E2, but not the alpha enantiomer, inhibits basal and interleukin-1 beta-stimulated expression of ICAM-1 as well as NF-kappaB activation in immortalized rat brain endothelial cells [123]. VCAM-1 is an important molecule, mediating mononuclear cell adhesion to endothelial cells. In ECV 304 cells transfected with an ER alpha expression plasmid to overexpress this receptor, E2 inhibited the activating effects of TNF-alpha on NF-kappaB activation, VCAM-1 expression, and adhesion of monocytes. These findings suggested that E2 can suppress inflammatory cell adhesion to vascular endothelial cells that possess functional ES receptors [124]. E2 also decreases VCAM-1 expression by an inhibitory effect at the level of the transcription factors NF-kappaB, AP-1, and GATA. This was observed in LPS-induced VCAM-1 expression in human vascular endothelial cells [125]. IL1-beta also induced VCAM-1 expression in HUVECs and this effect could be suppressed by preincubation with E2 (250 pg ml–1 or 500 pg ml–1) [126]. 17-Epiestrol, an estrogen metabolite, is more potent than 17-beta E2 in suppressing TNF-alpha-induced VCAM-1 expression, an action modulated at least in part through nitric oxide [127]. These results are summarized in Table 13.3.

13.3.4 Estrogens and Experimental Autoimmune Encephalomyelitis

ES treatment has been shown to protect against EAE, a disease considered as an animal model of MS [128]. Such an effect was first observed after a longterm treatment of EAE in mice with high doses of hormone [129]. More recent data suggest an effect of low-dose therapy: diestrus levels (< 100 pg ml–1 in serum) of E2 significantly reduce the clinical manifestation of active EAE in both

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Table 13.3 Adhesion molecules expressed in endothelial cells (ECV 304: human endothelial-like cells; HBECs: human brain endothelial cells; HUVECs: human umbilical vein endothelial cells; HSVECs: human saphenous vein endothelial cells) and involved in leukocyte recruitment at the BBB. Effects of ES. Name of cell or cell line

Name of gene

Description

Inducers of gene expression

ES used (effects on gene expression)

Ref.

HBECs, HUVECs

E-selectin, ELAM-1

TNF-alpha, interleukin-1beta

Integrins (a5, a6, b1) ICAM-1 (CD54)

Intercellular adhesion molecule Vascular cell adhesion molecule Vascular cell adhesion molecule Vascular cell adhesion molecule Vascular cell adhesion molecule Vascular cell adhesion molecule

Interleukin-1beta

E2, 17betaestradiol (downregulation) E2 (upregulation) E2 (downregulation) E2 (upregulation ) E2 (downregulation) 17-epiestrol (downregulation) E2 (downregulation) E2 (downregulation)

109, 117

HUVECs

Selectin, endothelial leukocyte adhesion molecule Integrins

HUVECs HUVECs ECV 304 HUVECs HUVECs HSVECs

VCAM-1 (CD106) VCAM-1 (CD106) VCAM-1 (CD106) VCAM-1 (CD106) VCAM-1 (CD106)

no induction

TNF-alpha (24 hours exposure) TNF-alpha TNF-alpha Interleukin-1beta LPS

122 109, 117, 124 109 124 127 117, 123 120

male and female mice [130]. In addition, the mechanism of E2 protection appears to involve both systemic inhibition of TNF-alpha expression and local recruitment of inflammatory cells into the CNS of mice developing EAE [131]. A recent study demonstrated that, although ES is generally assumed to act by modulating immune functions, E2 treatment can inhibit EAE without affecting autoantigen-specific T cell responsiveness and type 1 cytokine production. In fact, the beneficial effect of E2 does not involve ER-alpha signaling in blood-derived cells. These authors suggest a role for ER-alpha expressed in CNS-resident microglia or endothelial cells in the mediation of this protective effect [132]. Nonlymphocytic cells, such as macrophages, dendritic cells, or other nonlymphocytic cells, were suggested to be primarily responsive to E2 treatment in EAE [133]. DNA microarrays have been used to evaluate the effects of E2 on gene expression in EAE and MS. In the case of EAE, E2 treatment affected about 10% of the genes tested, and only 18 cytokine, chemokine/receptor, adhesion molecule, or activation genes were up- or downregulated more than 2.4-fold by this treatment. Thus, it was clearly shown by this study that the ES effect is restricted to several specific genes. In mice splenocytes, CTLA-4 (known to inhibit T cell activation), two interferon gamma-induced genes, TGF-beta3, IL-18, chemokines, VCAM-1, and a disintegrin metalloprotease (thought to regulate TNF-alpha pro-

13.4 Conclusions and Perpectives

duction) were found to be upregulated. In contrast, TNF-alpha, an important proinflammatory cytokine in EAE and MS, RANTES (known to be increased in EAE and MS), and NCAM (increased in cerebrospinal fluid from patients with active MS) were downregulated. In conclusion, this set of unexpected E2-sensitive genes may be of interest for the development of novel strategies for the treatment of EAE and possibly MS [134]. The hormone estriol, which is elevated during pregnancy, has been shown to alleviate EAE when administrated in vivo at these levels [135]. Recently, a pilot clinical trial using oral estriol was completed and relapsing-remitting MS patients demonstrated significantly decreased gadolinium enhancing lesions [136]. Accordingly, the immunomodulatory effects of oral estriol therapy were assessed and significant increases in the levels of IL-5 and IL-10 and a decrease in TNF-alpha were observed in stimulated peripheral blood mononuclear cells [137]. New genes involved in MS were also found: increased transcripts of genes encoding inflammatory cytokines, particularly IL-6 and IL-17, interferon-gamma, and associated downstream pathways, were observed by microarray analysis of MS lesions [138]. Some products of these genes have been chosen as targets for EAE therapy in mice. For example, when granulocyte colony-stimulating factor (G-CSF) was given before the onset of EAE, its influence is essentially exerted on the acute rather than the chronic stage of the disease. The reversal of EAE with G-CSF has been reported [139]. The EAE result is corroborated with the transcriptional analysis of active and silent MS lesions: G-CSF is upregulated in acute, but not in chronic MS lesions and the effect on EAE is more pronounced in the acute phase of the disease [138].

13.4 Conclusions and Perspectives

The activated cerebrovascular endothelium is an important target for both GC and ES. By regulating the expression of genes involved in leukocyte-endothelial cell interactions, these hormones can play a crucial role in the modulation of recruitment of activated leukocytes during chronic inflammatory diseases. In addition, this activated endothelium is an attractive target for pharmacological intervention in order to inhibit endothelial cell activation as well as the consequent recruitment of activated leukocytes in order to improve the therapy of such diseases. GC are commonly used in anti-inflammatory therapy. In particular, acute relapses in MS are treated with GC. GC-Dependent suppression of the expression of the genes coding CAM is now an accepted mechanism of action of these hormones. In the case of ICAM-1 and ICAM-2, which have been identified as essential regulators of transendothelial migration of autoaggressive T cells, GC can modulate CAM expression by the repression of NF-kappaB signaling [58, 61]. Elevated levels of ICAM-1 have been described in MS during relapses [140], and conversely, so have ICAM-1 decreases in MS during remission or following treatment with high-dose GC therapy [141]. However, long-term therapies using

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GC are usually accompanied by many severe side-effects: atrophy of the skin, myopathy, osteoporosis, and psychosis. To improve this therapy, new GR ligands have been synthesized with highly potent immunosuppressive and anti-inflammatory properties, but reduced side-effects. These new molecules (dissociated GC) have been shown to possess anti-inflammatory and immunosuppressive activity as potent as the glucocorticoid prednisolone in two in vivo models. These dissociated GC strongly inhibit AP-1, but have little or no transactivating activity. These results have provided a novel concept for drug discovery [142]. Another possibility for the selective inhibition of endothelial cell activation is the development of selective immunoconjugates like the DEX-anti-E-selectin immunoconjugate, which is successfully internalized by activated endothelial cells [98, 99]. The intercellularly delivered DEX is then fully able to exert its pleiotropic anti-inflammatory activity [143]. In the future, analogous strategies may also prove useful for delivering dissociated GCs to inhibit endothelial cell activation.

Acknowledgment

I thank Dr. Nancy Grant for critical reading of the manuscript.

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14 Metalloproteinases and the Brain Microvasculature Dorothee Krause and Christina Lohmann

14.1 Introduction

Endothelial cells, glial cells, pericytes and extracellular matrix components are collectively involved in the process of interactive blood-brain barrier (BBB) regulation and modulation in response to environmental stimuli [1]. In addition to the structural components of the BBB, a complex set of specific transport systems such as glucose transporters, certain amino acid transporters, and transferrin receptors provide directed transport of nutrients across the BBB into the central nervous system. Additional transporter molecules serve a protective function, to keep toxic metabolites out of the brain, such as P-glycoprotein (see Chapter 19). Moreover, the cerebral microvasculature contains a battery of enzymes which serve complementary functions in maintaining the metabolic properties of the BBB complex. Some of these enzymes are able to degrade a wide variety of peptides at or inside the vascular wall and thus constitute an “enzymatic barrier” to the passage of peptides across the BBB; others are able to degrade extracellular matrix molecules. These enzymes are proteinases/proteases and peptidases (both terms are often used as synonyms) which have been detected at the BBB microvasculature of various species. Current knowledge on the in vivo function of proteinases/peptidases at the BBB is still limited. Possible roles are: (a) to prevent the entry of peripheral peptides into the central nervous system, (b) to modulate effects of circulating peptides on local blood flow, (c) to facilitate metabolism of locally released perivascular peptides, and (d) to remodel the extracellular matrix during angiogenesis and wound healing. Several studies have shown the involvement of BBB proteinases in the degradation of various peptides with vasoactive and neurotransmitter or neuromodulator properties, including monoamines, somatostatin, enkephalins, angiotensin I and II, kallidin, bradykinin, and somatostatin analogues [2].


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14 Metalloproteinases and the Brain Microvasculature

14.2 Metalloproteinases in Brain Microvessels: Types and Functions

Metalloproteinases (MPs), often referred to as metalloproteases or metallopeptidases, are proteolytic enzymes which use a metal ion for their catalytic activities. Extracellular MPs are crucial for some cell functions entailing complex roles at the cell surface. MPs located at the BBB comprise aminopeptidase A (EC 3.4.11.7) [3], aminopeptidase N (EC 3.4.11.2, also known as aminopeptidase M) [4], carboxypeptidase N [5], angiotensin-converting enzyme (ACE, EC 3.4.15.1) [6], low levels of the neutral endopeptidase 24.11 (enkephalinase, NEP, EC 3.4.24.11) [7], the serine peptidases dipeptidyl peptidase II and IV (DPP) [2, 8], and matrix metalloproteinases [9] (see Table 14.1). Most of them have a catalytic domain with a zinc-binding motif, others contain a cobalt ion. The aminopeptidases are ectoenzymes, which cleave either the first or the second peptide bond within a protein to release the N-terminal amino acid which consists of a dipeptide. Generally they are Zn2+-dependent, although aminopeptidase A is activated by Ca2+. Aminopeptidase A, N(M), and W have been detected in isolated brain microvessels. Aminopeptidase A is suggested to be involved in cerebrovascular metabolism of angiotensins whereas aminopeptidase N(M) has been particularly implicated in the inactivation of enkephalins [3]. Bestatin has been shown to be a slow-binding, competitive inhibitor for most aminopeptidases [10]. The neutral endopeptidase 24.11 (enkephalinase) also cleaves enkephalins.

Table 14.1 Localization of metalloproteinases at the cerebral microvasculature. Type

Synonym

Location at the BBB

Aminopeptidase A

Glutamyl-aminopeptidase (EC 3.4.11.7)

Pericytes

Aminopeptidase N(M)

Alanin aminopeptidase (EC 3.4.11.2)

Pericytes, microglia, (astrocytes?, endothelium?)

Aminopeptidase W Angiotensin-converting enzyme (ACE)

Cerebral microvasculature Peptidyl dipeptidase A (EC 3.4.15.1)

Endothelium

Endopeptidase 24.11

Enkephalinase

Cerebral microvasculature, pericytes

Dipeptidylpeptidase IV (DPP IV)

Serine peptidase IV

Endothelium

Dipeptidylpeptidase II (DPP II)

Serine peptidase II

Cerebral microvasculature

Matrix metalloproteinases (MMPs ; see Table 14.2)

Matrixins

Endothelial cells, pericytes, astrocytes, microglia, neurons

Carboxypeptidase N

Endothelium


The angiotensin-converting enzyme (ACE) is a Zn2+-dependent metalloproteinase activated by Cl– ions. It hydrolyzes dipeptides from the C-terminus and converts angiotensin I to the active vasoconstrictor angiotensin II. It also inactivates the vasodilatator bradykinin. Both angiotensin II and bradykinin not only constitute vasoactive substances, but also act as neurotransmitters. ACE may therefore have a vital role in the regulation of cerebral blood pressure and in peptide metabolism. Similar to ACE, the metalloproteinase carboxypeptidase N was found to cleave the kinins kallidin and bradykinin and to degrade vasopressin into fragments. The dipeptidylpeptidases (DPP) II and IV are serine peptidases that degrade small peptides and proteins containing proline residues (e.g. substance P) which is resistant to degradation by aminopeptidase N(M) [2]. One larger family of metalloproteinases identified in cerebral microvessels with zinc-dependent proteolytic activity are the matrix metalloproteinases (MMPs), which under normal and pathological conditions possess complex functions at the cell surface and within the extracellular matrix of the central nervous system. MMPs are able to degrade virtually all types of extracellular matrix components and are known to be responsible for the maintenance, turnover, and integrity of the extracellular matrix, suggesting they play a central role in several biological processes such as embryogenesis, organogenesis, wound healing, angiogenesis, and in multiple steps of inflammation. Under normal physiological conditions, MMP expression and activity are precisely balanced at different levels including: (a) the level of transcription and translation, (b) activation of latent pro-enzymes by other proteinases and free radicals, and (c) inhibition by endogenous tissue inhibitors of metalloproteinases (TIMPs) and a-macroglobulins. Several synthetic peptides have also been shown to be inhibitors of MMP activities [11, 12]. TIMPs are specific MMP inhibitors that participate in controlling the local activities of MMPs in tissues by binding to the catalytic site of MMPs. Four TIMPs (TIMP-1, -2, -3, -4) have been identified in vertebrates. Their expression is differentially regulated during ontogenic development and tissue remodeling. Changes of TIMP levels are considered to be clinically important because they directly affect the level of local MMP activities; and a loss of MMP control may result in pathological conditions. When the delicate balance between the production and proteolytic activity of MMPs on the one hand and their inhibition by TIMPs on the other hand is disturbed towards increased MMP activity, uncontrolled breakdown and widespread destruction of the extracellular matrix occurs. Conversely, excessive expression of TIMPs leads to the restriction of physiological proteolysis and a net buildup of extracellular matrix proteins, resulting in fibrosis. In the process of neovascularization, the key role of MMPs and TIMPs is the control of dysregulated vascular growth which is characteristic for a number of angiogenic diseases. Twenty-eight MMP genes have been identified so far, of which 23 are found in humans (see Table 14.2). Most of them are multidomain proteins which are either secreted by the cell into the extracellular space or anchored to the plasma membrane [13].

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14 Metalloproteinases and the Brain Microvasculature Table 14.2 Matrix metalloproteinase family members. Matrix metalloproteinase

Function

Collagenases

Collagenolytic activity, digestion of ECM and non-ECM molecules

MMP-1 (collagenase-1) MMP-8 (collagenase-2) MMP-13 (collagenase-3) MMP-18 (collagenase-4, identified in Xenopus) Stromelysins MMP-3 (stromelysin-1)

Activation of pro-MMP-9, expressed in endothelial cells

MMP-10 (stromelysin-2) MMP-11 (stromelysin-3) Matrilysins MMP-7 (matrilysin-1) MMP-26 (matrilysin-2, endometase) Gelatinases MMP-2 (gelatinase-A) MMP-9 (gelatinase-B)

Membrane-type MMPs MMP-14 (MT1-MMP) MMP-15 (MT2-MMP) MMP-16 (MT3-MMP) MMP-17 (MT4-MMP) MMP-24 (MT5-MMP) MMP-25 (MT6-MMP) Other MMPs MMP-12 (metalloelastase) MMP-19 MMP-20 (enamelysin) MMP-22 = MMP-21 MMP-23 MMP-28 (epilysin)

Cleave gelatine, fibronectin, elastin and different collagens Expressed in astrocytes, Schwann cells, and peripheral nerves Expressed in brain endothelial cells, astrocytes, pericytes, and microglia; markedly upregulated in inflammation Activation of pro-MMP-2

Macrophage migration; mainly expressed by alveolar macrophages Autoantigen from patients with rheumatoid arthritis Digestion of amelogenin; exclusively expressed by odontoblasts

Wound repair


MMPs, collectively called matrixins, can be divided into six subgroups, depending on their substrate specificity, sequence similarity and domain organisation [14, 15]. Transcription of many MMPs is promoted by inflammatory cytokines, growth factors, chemokines, oncogenes, and cell-cell or cell-matrix interactions. Further, it is known that MMPs are intracellularly synthesized as latent enzymes (proMMPs) and most pro-MMPs are activated extracellularly. The activation process is a stepwise mechanism which can be triggered either by proteinases in vivo or by chemical agents, low pH, and heat treatment, as shown in vitro. Activation factors include parts of the plasminogen-plasmin cascade [16], as well as other MMPs which proteolytically cleave the propeptide region of effectors which disrupt the interaction between cysteine and zinc (the so-called “cysteine switch” mechanism) to trigger activation [17]. A few MMPs are processed at the cell membrane into fully active enzymes [18]. Active MMPs are differentially inhibited by the binding of TIMPs. TIMP-1 inhibits MMP-1, MMP-3 and MMP-9 more effectively than TIMP-2. TIMP-3 inhibits MMP-2 and MMP-9 [19], whereas TIMP-4 is a good inhibitor of all classes of MMPs without remarkable preference [20]. Interestingly, TIMPs are also required for the activation of some MMPs [21]. These complex mechanisms are described exemplarily for the cell surface activation of MMP-2 through a membrane-type MMP (MT-MMP)-mediated cascade. MT-MMPs are membrane proteins which are either glycosyl-phosphatidylinositol-anchored or transmembrane proteins. They frequently function as activating proteins, e.g. MT1-MMP (MMP-14) has been found to bind to a complex formed by TIMP-2 and proMMP-2 to facilitate the activation of MMP-2 [22]. TIMP-2 mediates pro-MMP-2 activation in a dose-dependent manner. Pro-MMP-2 is processed at low TIMP-2 concentrations, but inhibited at higher TIMP-2 concentrations [23]. MT2-MMP may activate pro-MMP-2 through a TIMP-2-independent route [24]. MMP-2 in concert with MT1-MMP can activate pro-MMP-13. Two recently discovered new families of MMPs also appear to play an important role in brain function, the ADAMs (a disintegrin and metalloproteinase) [25] and the ADAMTs (a disintegrin and metalloproteinase with a thrombospondin motif) [26]. ADAMs are with few exceptions transmembrane proteins, while the ADAMTs are secreted molecules, some of which bind to the extracellular matrix [27]. These enzymes cleave a number of extracellular matrix molecules and remove ectodomain molecules from the cell surface, such as TNF-a receptor, interleukin-6, L-selectin and syndecans [28]. Literature on the role of ADAMs and ADAMTs in the BBB is still sparse and little is known about the regulation of their activity.

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14.3 Cerebral Endothelial Cells and Metalloproteinases

MMPs are able to cleave the basal lamina macromolecules that line the BBB microvessels, leading to disruption of the BBB and modulation of capillary permeability [9]. However, it is to date poorly understood if and how MMPs are involved in the regulation of BBB function. Regulation of paracellular BBB permeability is a complex process involving the intracellular signaling and rearrangement of tight junction proteins in cerebral endothelial cells. Several studies have reported markedly increased BBB permeability after exposure to TNF-a, interleukin-1b, interferon-c, histamine, and growth factors. It is known from in vivo studies that intracerebral MMP injection leads to extensive leakage of the BBB and T-cell recruitment to the lesion site and demyelination. This lesion could be reduced by MMP inhibitors [29]. In vitro investigations by Lohmann et al. [30] show that MMP-2, present in serum, decreases the transendothelial electrical resistance of cultured porcine brain endothelial cells. In the same model, the glucocorticoid hydrocortisone suppresses the secretion of MMPs by brain endothelial cells and simultaneously results in improved barrier function. Moreover, proinflammatory cytokines and chemokines, which markedly decrease the barrier function of cerebral endothelial cells, have been shown to regulate the secretion and activation of MMPs [31], suggesting MMPs to be the missing link in BBB permeability regulation by cytokines. Thus, clear evidence is given for the role of MMPs in endothelial tight junction regulation. However, the molecular mechanism of action of MMPs on the tight junction complex remains to be elucidated. Lohmann et al. [30] investigated the regulatory mechanisms of tight junction permeability and MMP interaction in a well characterized in vitro model of the BBB, based on primary cultured pig brain capillary endothelial cells of a polarized phenotype which displays a tight and intact cellular barrier [32]. The tightness of the cellular BBB in vitro can be directly measured as transendothelial electrical resistance (TEER, see Chapter 16), reflecting the permeability of the tight junctions for small ions and solutes [33]. Zymographic analysis revealed that pig brain endothelial cells secrete MMP-9 apically (to the blood side in vivo). Lohmann et al. [30] show that secretion of MMP-9 is markedly reduced after the addition of hydrocortisone (550 nM) to the culture medium, which at the same time results in improved barrier function. Fluorimetric analysis of enzymatic activity revealed that hydrocortisone concomitantly reduces MMP activity at the BBB in vitro. A basolateral (to the brain side in vivo) secretion of MMP-9 by cerebral endothelial cells in vitro was also demonstrated by immunocytochemistry, where MMP-9 was found to be attached to the extracellular matrix. In addition, endogenous MMP-inhibitors in form of TIMP-1 and TIMP-2 were detected in an in vitro BBB model by immunocytochemistry and Western blot analysis. The studies of Herron et al. [34, 35] describe the production of pro-MMP-1 and proMMP-3 in rabbit cerebral endothelial cell cultures. These authors observed that

14.3 Cerebral Endothelial Cells and Metalloproteinases

the absence of enzymatic activity is due to the simultaneous production of inhibitors. The inhibitors are secreted by endothelial cells into the medium and are at least partially bound to MMPs in the extracellular matrix. In this way cerebral endothelial cells in vitro seem to balance their proteolytic side under these conditions. MMP activity at the BBB in vitro can be significantly increased by addition of the tyrosine phosphatase inhibitor phenylarsine oxide (PAO) in a time- and dose-dependent fashion [30]. Increased MMP activity was paralleled by a significant decrease in TEER, indicating impaired barrier function, and morphological changes, implying the disruption of cell-cell contacts and the formation of intercellular gaps. Moreover, Western blot analysis of tight junction proteins revealed PAO-induced proteolysis of occludin, with a generated fragment of approximately 51 kDa [30, 36], but the tight junction proteins zonula occludens-1 (ZO1) and claudin-5 remained intact. Tyrosine phosphatase inhibition by pervanadate without concomitant MMP activation did not induce occludin proteolysis. Occludin has previously been shown to be a substrate for serine proteases [37]. As occludin proteolysis at the BBB in vitro could specifically be prevented by MMP inhibitors, GM 6001, zinc ions and 1,10-phenanthroline [30, 36], there is now direct evidence for increased MMP activity upon exposing the cells to PAO, leading to occludin proteolysis and loss of barrier integrity. The described data give direct evidence of an involvement of MMPs in the regulation of BBB function in vitro and suggest that cleavage of the tight junction protein occludin by MMPs is a possible molecular mechanism of BBB regulation. Figure 14.1 schematically depicts the described activation pathways of MMPs in cerebral endothelial cells. It appears that increased MMP activity at the BBB leads to MMP-dependent cleavage of the tight junction protein occludin and a profound disruption of cell-cell contacts, resulting in markedly compromised barrier integrity and function. Additionally, Asahi et al. [38] reported that MMP-9 can proteolytically cleave ZO-1. Thus, extracellular activity of MMPs could be a crucial and site-restricted determinant of the unique properties of BBB interendothelial tight junctions. Comparable results were shown recently in diabetic retinopathy, which involves the breakdown of endothelial tight junctions of the blood-retinal barrier (see Chapter 26). The retinas of diabetic animals demonstrated elevated levels of MMP-2, MMP-9, and MMP-14 mRNA, accompanied by an increase in vascular permeability and a proteolytic degradation of the tight junction protein occludin [39]. Behzadian et al. [40] demonstrated that retinal endothelial cells express MMP-9 when treated with TGF-b or when cocultured with glial cells (astrocytes or Muller glial cells) and that both TGF-b and MMP-9 increase retinal endothelial cell permeability. This mechanism may also contribute to the breakdown of the blood-retinal barrier. Miyamori et al. [41] could identify the tight junction protein claudin 5 as one factor which promotes the activation of pro-MMP-2 mediated by MT1-MMP in 293 T cell cultures. These results suggest a more dynamic function for claudins than simply being structural constituents of tight junctions; and they also indi-

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Fig. 14.1 Possible mechanism of BBB permeability regulation by MMPs. Cerebral endothelial cells secrete pro-MMPs. Pro-MMPs are proteolytically activated by other active MMPs or by cell membrane-bound active membrane-type MT-MMPs. In a next step, active MMPs proteolytically cleave the tight junction protein occludin within the first

extracellular loop near the N-terminus [30]. This has a destabilizing effect on cell-cell contacts and results in increased transendothelial permeability. In this context, phenylarsenoid (PAO) has a feasible effect on extracellular MMP activity, either by increasing the expression of membrane-bound MT-MMP or by binding pro-MMPs to MT-MMPs.

cate a precisely located effect of MMPs at the tight junction complex. These regulatory processes are supplemented by novel findings on the roles of the low density lipoprotein (LDL) receptor family which show that LDL receptor-related protein (LRP) interacts as a regulator of vascular tone, permeability of the BBB, and expression of MMPs [42]. Besides the described endothelial expression of MMP-9, it is known that astrocytes, oligodendrocytes, microglia, and neurons can also secrete MMP-9 [43].

14.4 Perivascular Cells and Metalloproteinases

14.4 Perivascular Cells and Metalloproteinases 14.4.1 Pericytes

A predominant role within the BBB is played by the cells closest to the cerebral endothelium, the perivascular cells (“pericytes”, see Chapter 5). Pericytes are positioned along the vessel axis, forming numerous cytoplasmic processes that encircle endothelial cells. However, they are separated from the cerebral endothelium by a common basement membrane. They take up blood-borne substances through endo- and phagocytosis and they are recognized as immunocompetent antigen-presenting cells, which participate in angiogenesis. Furthermore, they are suggested to play a regulatory function in the control of capillary growth and tube formation [44]. These findings provide evidence that cerebral pericytes act as a “second line of defense” to maintain metabolic homeostasis between blood and brain [45]. Balabanov and Dore-Duffy [46] postulated that pericytes constitute an important component of the BBB, with an involvement in virtually all processes at the BBB. However, the involvement of pericytes in BBB regulation is still a matter of discussion (see Chapter 5). Immunocytochemical studies have shown the existence of BBB-specific enzymes in pericytes, e.g. c-glutamyltranspeptidase (c-GT) [47], alkaline phosphatase, and the metalloproteinases aminopeptidase A [48, 49], and aminopeptidase N(M) (APN) [45, 50]. Aminopeptidase A (APA) is the only enzyme so far discovered in the cerebral microvasculature that degrades angiotensin II to angiotensin III [3]. APA shows highest expression in brain regions that lack a tight BBB endothelium, such as the median eminence and choroid plexuses [48], whereas aminopeptidase N is confined to sites of tight BBB microvessels. The abundance of aminopeptidase A at the circumventricular organs correlates well with the fact that these brain regions are highly enriched in angiotensin II receptors [51]. To date, only APN has been clearly characterized and its ultrastructural localization as an extracytoplasmic membrane-bound enzyme of cerebral pericytes (pericytic aminopeptidase N; “pAPN”) has been described [45, 50]. APN was first reported by Solhonne et al. [52] as a membrane-bound ectopeptidase with a broad activity for almost all unsubstituted oligopeptides and is involved in the extracellular degradation and inactivation of endogenous opioid peptides, e.g. enkephalines and endorphines. pAPN is not expressed at the endothelial side of the BBB frontier. Microvasculature of brain regions that lack BBB properties, such as the area postrema, median eminence, and choroid plexuses, are devoid of pAPN expression. pAPN is asymmetrically distributed at the pericytic plasma membrane with preference at the abendothelial sites. At the pericytic cell processes, this asymmetric distribution is less evident. The predominant localization of pAPN at the parenchymal front rather than at the vascular site indicates a primarily parenchymal control function of pAPN. Thus, endogenous unsubsti-

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tuted peptides can be degraded easily before entering the periendothelial space. In contrast, the lack of pAPN in the circumventricular organs allows for the exchange of endogenous peptides from blood to brain and vice versa. The onset of pAPN in embryonic rat brain is known to occur on day E18. A steady-state level is reached at postnatal day 6–8 [50]. The exclusive expression of pAPN at cerebral pericytes indicates an important metabolic function within the BBB control mechanisms. In this context, primary cultured endothelial cells as well as cocultures with astrocytes and pericytes are useful in vitro alternatives to investigate blood vessels in vivo for studying the BBB function. Pericytes are also involved in specific microvascular diseases and in angiogenesis. In cocultures with endothelial cells, pericytes secrete an active form of TGF-b that controls endothelial growth and appears to mediate pericyte-endothelium interactions, like guiding the migrating endothelial cells and forming connections between newly formed sprouts [44, 53, 54]. Angiotensin II can contribute to the regulation of retinal neovascularization by stimulating the migration of pericytes in retinal microvessels, augmenting latent MMP-2 activity, and doubling the TIMPs activity [55]. Pericytic angiotensin II-induced chemotaxis is mediated by antibodies against platelet-derived growth factor (PDGF) and involves TGF-b. Chantrain et al. [56] demonstrate that MMP-9 in neuroblastoma contributes to angiogenesis by promoting blood vessel morphogenesis with pericyte recruitment. Girolamo et al. [57], using human developing brain microvascular cultures, show the concerted interplay between endothelial cells and pericytes in angiogenesis regulation by expression of MMP-2. Endothelial cells express pro-MMP2 and pericytes express active MMP-2 [57]. The active MMP-2-containing pericytes may be considered as cells cooperating with the endothelial cells to bring about basal lamina degradation and play a role as “pioneers” that dissect the brain parenchyma and allow endothelial sprouting during angiogenesis. Together, these expression patterns highlight the enzymatic and metabolic aspect of the pericytic barrier function.

14.4.2 Astrocytes and Microglia

Astrocyte endfeet cover much of the capillary’s basal surface and they are known to induce BBB formation [58]. They participate in a variety of homeostatic functions and are involved in mechanisms of neural injury and repair (see Chapters 8 and 9). Furthermore, astrocytes induce the differentiation of cerebral capillary endothelial cells into cells with BBB characteristics; and this induction is mediated by soluble products released by the astrocytes [59]. Metalloproteinase expression is also described in astrocytes, but most of the investigations of MMP expression in astrocytes have been done under cell culture condi-

14.4 Perivascular Cells and Metalloproteinases

tions. For example, Apodaca et al. [60] showed the presence of MMP-9 and MMP-2 as well as the inhibitors of these MMPs in conditioned cell culture medium from fetal astrocytes stimulated with phorbol esters. Similar observations were done by Gottschall and Yu [61] when astrocyte cultures were stimulated with LPS, IL-1b, or TNF-a. Under these conditions, astrocytes secreted MMP-1, MMP-2, and pro-MMP-9. MMP-1 has been shown to be toxic to human neurons in culture [62]. LPS and IL-1b also stimulate the production of MMP-3 in astrocytes [43]. However, astrocytes and microglia in coculture produce an active form of MMP-9 when stimulated by LPS [63]. Leveque et al. [64] reported on the effect of HIV infection and TNF-a on the expression of MMP-2 and MMP-9 in astrocytes. HIV infection increases the production of pro-MMP-2 and pro-MMP-9, suggesting that astrocyte-HIV contact may lead to extracellular matrix activation. Primary mouse astrocytic cultures stimulated with various cytokines and cellular growth factors (IL1-b, TNF-a, EGF) display increased levels of MMP-3 mRNA. Interferon-c inhibits this response. The mRNA accumulation was preceeded by activation of the transcription factors NFjB and AP-1, but this effect might not solely be responsible for the cytokine-induced expression of MMP-3 mRNA in astrocytes [65]. Further factors influencing the expression of MMPs in astrocytes include bradykinin and the heat-shock protein HSP 70. Bradykinin, an inflammatory mediator, has been shown to induce the expression of MMP-9 in rat astrocytes; and activation of p42/p44 mitogen-activated protein kinase (MAPK) mediated through NFjB pathways is essential for bradykinin-induced MMP-9 gene expression [66]. In contrast, HSP 70, which is synthesized in response to a variety of stress factors including ischemia, suppresses MMP-2 and MMP-9 production in astrocytes [67]. These findings suggest that HSP 70 may play a protective role by down-regulating MMPs in stress situations. Another key homeostatic mechanism of astrocytes in tissue repair is maintained through their production of TIMP-1. Gardner and Ghorpade [68] showed astrocytic TIMPs expression in inflammatory neurodegenerative diseases, which may have significant therapeutic relevance. Colton et al. [69] showed the production of MMP-9 in activated microglial cells. Microglia activation reveals a biphasic process: an early phase of global activation is followed by a later phase in which microglia activation becomes increasingly focused in the lesions. During the early phase, expression of pro-inflammatory mediators like IL-1b TNF-a, and early growth response-1 (Erg-1) increases but is restricted to lesions [70]. Immunostaining of astrocytic and microglial cocultures showed the expression of MMP-3 by microglial cells but not by astrocytes. Further findings suggest that MMP-3 produced in microglia is important for the activation of MMP-9 produced in astrocytes during LPS stimulation [9]. Aminopeptidase N is also expressed in microglial cells. However, it is not a stable marker for microglia because its expression apparently is related to functional or morphological changes of microglia [71].

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Fig. 14.2 Schematic view of the interaction of metalloproteinases (MPs) at the bloodbrain barrier (BBB). Under normal conditions, there is a constitutive expression of pro-MMP-2 by astrocytic endfeet with the potential for activation by the membrane-type MMP (MT-MMP). Pericytes express aminopeptidase N and A. During cellular stress, some other MPs come into action. MMP-9 is secreted by lymphocytes and endothelial cells

damaging the endothelial barrier and the basement membrane. Pericytes and microglial cells secrete pro-MMP-2, pro-MMP-3, and pro-MMP-9. Activation of pro-MMP-3 may be facilitated by the plasminogen/plasmin system in pericytic/microglial membranes. Possibly, activated MMP-3 could activate pro-MMP-9. This cumulative activation of MMPs could amplify BBB impairment and cause further brain dysfunction.

All in all, these data indicate that astrocytes, besides their physiological production of MMP-2, could be a major source of MMP-9 in the developing and inflamed brain. Thus, cytokines appear to be potent regulators of MMP production in astrocytes and microglia during inflammation. A model of the distribution of metalloproteinases in brain capillaries is given in Fig. 14.2.

14.5 Metalloproteinases and the Blood-Liquor Barrier

The epithelial cells of the choroid plexus form the structural basis of the barrier between the blood and cerebrospinal fluid. They are involved in transport processes from blood to cerebrospinal fluid (CSF) and vice versa. Expression of various metallopeptidases has been detected at the blood-CSF barrier [72]. Strazielle et al. [73] demonstrated that the choroid plexus is a source of pro- and active MMP-2 and MMP-9 in the brain and that pro-inflammatory cytokine treatment leads to an increase in choroidal MMP secretion at either the apical or basolat-

14.6 Metalloproteinases and Brain Diseases

eral membrane. Thus, during inflammation, the choroid plexuses could comprise a source of MMPs found in the cerebrospinal fluid, which facilitates leukocyte migration. Concerning the distribution of further MPs, Schnabel et al. [74] showed a reduced activity of dipeptidylpeptidase IV, low activity of aminopeptidase A, and no activity of gamma-glutamyl transpeptidase in capillaries of the choroid plexuses. These authors described the presence of aminopeptidase M in leaky capillaries, but these findings were not substantiated by others [50].


Blood-brain barrier disruption is common in many neurological diseases. Under pathological conditions the barrier becomes compromised, resulting in an intense cell trafficking from blood to brain, and the central nervous system becomes enriched with immune competent cells. Activated cytotoxic lymphocytes [75], macrophages, or certain types of metastatic cells [76] can even cross the intact BBB. In a first step, these cells obviously share the ability to recognize and bind to endothelial cells. Less clear in this context is the entrance mechanism by which these cells cross the intact BBB and how they manage to enter the brain parenchyma (see Chapters 4 and 18). A better understanding of these processes is needed to design therapeutic strategies with the aim to prevent monocyte infiltration across the BBB. Some investigators have claimed that the first transmigration step occurs transcellularly through endothelial cells [77], while subsequent excessive infiltration of monocytes by paracellular movement is accompanied by endothelial damage, resulting in the loss of tight junctions [75, 78]. In a further step, these cells can affect the function of neurovascular structures by attacking the basal lamina around the cerebral vessels, leading to hemorrhage and cerebral edema. Neuropathogenesis of HIV-1 infection demonstrates a facilitated transmigration of HIV-infected monocytes/macrophages by an increased production of MMP-9 activity. Furthermore, chemokines regulate the traffic of the infiltrated monocytes through the brain parenchyma [79]. Much has been learned recently about the role of MPs in neurological diseases, but our understanding of their multiple roles is still incomplete. Particularly, most MMPs are largely absent from the normal BBB and central nervous tissue. However, their upregulation has been reported in several neurological disorders, with an increase in capillary permeability and leukocyte migration into the central nervous system. Changes in the fine tuning of MMPs expression and TIMPs seem to affect extracellular matrix turnover and has been implicated in pathological and neuroinflammatory conditions of the central nervous system, including meningitis, encephalitis, and brain tumors (see Chapter 24), AIDS dementia and multiple sclerosis (MS). Other proteases are also increased at sites of secondary injury, e.g. plasminogen activators (PAs), and may act in concert to attack the extracellular matrix [80].

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Synthetic inhibitors of MMPs have been developed for the treatment of cancer and other MMP-related diseases. These hydroxamate-based compounds have been shown to reduce injury in experimental allergic encephalomyelitis (EAE; a mouse disease model of human MS), experimental allergic neuritis (EAN), cerebral ischemia, intracerebral hemorrhage, and viral and bacterial infections of the central nervous system [9]. A better understanding of MP expression under various pathological conditions of the central nervous system will allow for the use of MMP inhibitors in the treatment of brain disorders.

14.6.1 Metalloproteinases and Cerebral Ischemia

MMPs are important mediators under stroke conditions, since they show significant upregulation after ischemia. Chang et al. [81] showed that the matrix metalloproteinase MMP-2 participates in matrix degradation and disruption of the basal lamina of cerebral microvessels during focal cerebral ischemia. MMP-2 is induced in T cells bound to VCAM-1 on endothelial cells during inflammation when transmigration through the endothelial cell layer and basement membrane commences [82]. MMP-2 is secreted in a latent form (pro-MMP-2) and can be activated in a direct or indirect manner. Direct activators are the membrane-bound MT1-MMP and MT3-MMP, which are also constitutively expressed in brain, whereas plasminogen/plasmin system enzymes interact as indirect activators, such as urokinasetype plasminogen activator (uPA) and tissue-type plasminogen activator (t-PA). tPA also upregulates MMP-9 in vitro and in vivo. This response is mediated by the LDL receptor-related protein (LRP), which avidly binds tPA and possesses signaling properties [83]. Yepes et al. [84] demonstrated that, in the initial stages of cerebral ischemia, the opening of the BBB is mediated directly by tPA and that this activity is independent of MMP-9 but requires interaction with LRP. Furthermore, oxidative stress generated during stroke could mediate BBB disruption through local MMP-9 activation [85]. Asahi et al. [38], using knockout technology, showed that MMP-9 displays a central role in ischemic damage. MMP-9 knockout mice revealed smaller infarct volumes and exhibited protection against ischemic and traumatic brain injury. The protective effect of the MMP-9 gene knockout may be mediated by reduced proteolytic degradation of BBB components. These studies indicate that MMP-9 may play a critical role in the broad pathophysiology of cerebral ischemia. However, MMPs might also provide beneficial effects after injury in the central nervous system (e.g. axonal regrowth, remyelinization, angiogenesis) [22].


14.6.2 Metalloproteinases and Brain Tumors

Many MMPs were first cloned from tumors or tumor cell lines, suggesting that an excess of MMP activity is common in many brain tumors. Enhanced MMP expression in cancer often correlates with poor survival prognosis. MMP secretion facilitates migration and invasion of tumor cells through the extracellular matrix for penetration of the surrounding tissue [86]. High expression of metalloproteinases has also been demonstrated in brain tumor angiogenesis [87]. However, endothelial cells in brain tumors dedifferentiated and lose BBB properties [88].

14.6.3 Metalloproteinases and Multiple Sclerosis

MMPs seem to be implicated in multiple sclerosis (MS), where one of their suggested roles is to facilitate the transmigration of circulating leukocytes into the brain tissue and to disrupt the integrity of the BBB. Bar-Or et al. [89] investigated which of the 23 members of the human MMP family are critical to the MS disease process. They found a distinctive pattern of MMP expression in different cell populations, in which monocytes in particular show the strongest expression of different MMPs. This finding corresponds well with the rapid migration of monocytes across the BBB. Finally, the authors described higher levels of MMP-2 and MMP-14 in MS patients as compared to normal individuals. TIMP-2 levels were also elevated in monocytes from MS patients. The high expression of specific MMP members on monocytes can be used as a new target to design novel therapeutic strategies in MS. Maeda and Sobel [90] reported an increased expression of MMP-1, MMP-2, MMP-3, and MMP-9 by macrophages in acute MS and necrotic lesions, whereas chronic MS lesions revealed fewer MMP-positive macrophages. These data indicate that acute multiple sclerosis MMP-mediated proteolysis may contribute to the breakdown of the BBB and leukocyte migration into the central nervous system. Overall, a common elevation of MMP-9 and MMP-12 is found in MS and in mouse and rat EAE, the animal model of MS [22]. During the early stages of EAE, MMP-2, MMP-3, MMP-7, and MMP-9 are present at elevated levels in rat brain. In the course of acute EAE, Kunz et al. [91] demonstrated a dramatic downregulation of microvascular associated pAPN expression during the clinical peak of EAE. In addition, pAPN is transiently expressed by infiltrating macrophages and probably by a subpopulation of activated microglial cells in the white matter of lumbar spinal cord. Thus pAPN expression which under normal conditions is represented by pericytes is a sensitive marker for monitoring BBB damage.

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14.6.4 MMPs and Migraine

It has been debated whether MMPs are involved in migraine aura, because activation of MMPs leads to leakage of the BBB and allows potassium, nitric oxide, adenosine, and other products released by a cortical spreading depression to reach and sensitize the dural perivascular trigeminal afferents [92]. Additionally, cortical spreading depression (CSD), a self-propagating wave of neuronal and glial depolarization, which is implicated in disorders of neurovascular regulation such as migraine, causes prolonged MMP-9 activity. In this case, MMP-9 activation initiates a cascade that disrupts the BBB [93].

14.7 Conclusion

The complexity of the role of MPs and MMPs at the BBB in health and disease is evident. MMPs are involved in tissue remodeling and repair during development, in tissue damage during inflammation with breakdown of the extracellular matrix around the cerebral blood vessels, and in recovery from injury. The roles of MPs include the degradation of neuropeptides. The BBB components (endothelial cells, pericytes, astrocytes) are furnished with a specific set of MPs and MMPs, but the types and the stimuli that induce proteinase expression are different for the various cell types. MMPs are rapidly upregulated after nearly all types of central nervous system dysfunctions; and inhibition of MMPs has been shown to prevent the progression of many neurological diseases. A goal for the future could be to design strategies in which the inhibition of MMP activities may be used to counteract their malignant effects occurring under disease conditions.

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Part IV Culturing the Blood-Brain Barrier


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15 Modeling the Blood-Brain Barrier Roméo Cecchelli, Caroline Coisne, Lucie Dehouck, Florence Miller, Marie-Pierre Dehouck, Valérie Buée-Scherrer, and Bénédicte Dehouck

15.1 Introduction 15.1.1 In Vitro BBB Model Interests

The first notions and the discovery of the blood-brain barrier (BBB) were done in vivo by Ehrlich [1] and Goldman [2]. Since their work, in vivo studies have attempted to evaluate the exchanges between blood and brain parenchyma. Since the middle of the 19th century, in vitro approaches were developed with, first, the use of isolated brain capillaries [3–5]. As these vessels are metabolically active, they allow numerous studies regarding enzymatic activity [6, 7]. However, many reasons, such as the presence of remnants astrocyte foot processes [8] and pericytes, as well as the rapid and serious depletion of ATP in these isolated vessels [9], encouraged researchers to establish brain capillary endothelial cell culture [10, 11]. In culture, brain capillary endothelial cells form an endothelium-like monolayer and retain many of the BBB properties; and thus they can be used as in vitro BBB models that allow going into further cellular and molecular mechanisms. Indeed, from that time, in vitro BBB models have played an important role in the development of our current understanding on the BBB. As simple and dynamic experimental systems, they can still be considered valuable tools for future investigations, such as cerebral endothelium disturbances observed in CNS infections and brain-targeting drug delivery. This chapter focuses on some of the current methodologies for the establishment of in vitro BBB models, presenting the knowledge allowing “the culture of BBB”, meaning the establishment of well characterized in vitro BBB models.


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15 Modeling the Blood-Brain Barrier

15.1.2 The BBB: Brain Capillary Endothelial Cells and Brain Parenchyma Cells

Since the end of the 19th century, brain capillary endothelial cells have been known to be responsible for the limited exchanges between blood and brain parenchyma. They form an endothelium which can be distinguished from the other vascular beds by the presence of continuous tight junctions and the absence of fenestration or channels [12]. Both of these characteristics reduce the nonspecific transport of molecules across the BBB. The blood-brain exchanges involve specific carrier-mediated transport systems that facilitate the uptake of nutrients. Thus, BBB characteristics are crucial for the maintenance of brain homeostasis. Numerous studies reported that the brain endothelium phenotype is induced by surrounding brain cells, such as astrocytes, microglia, pericytes and neurons [13–19]. Although a few studies have shown that brain parenchyma cells are able to induce tight junction structures in aortic endothelial cells [20], the differentiation occurs only partially as compared to cerebral endothelial cells [21]. We have also shown the responsiveness of aortic endothelial cells to glial cell population influences [22]. However, it is worth mentioning that, in this study, glial cells had to be first cocultivated with brain capillary endothelial cells before being able to interact with aortic endothelial cells. As indicated in other studies, this suggests that specific bidirectional exchanges between brain parenchyma and brain endothelium are necessary for the establishment of BBB properties [15] and may explain why noncerebral endothelial cells are less responsive to brain parenchyma cells. From all these studies, the idea emerges that the cerebral endothelial phenotype might contribute to a better responsiveness to brain parenchyma cells and explain why starting from brain microvessel endothelium is more successful for recreating the in vivo BBB situation in vitro.

15.2 Culturing Brain Capillary Endothelial Cells 15.2.1 Brain Capillary Endothelial Cell Isolation 15.2.1.1 Brain Capillary Endothelial Cell Isolation Although everyone agrees that the BBB is localized at brain endothelium, a differentiation of endothelia derived from different parts of the brain should be done (Fig. 15.1). Indeed, a high level of heterogeneity in the properties of an endothelial cell population has been associated with the diverse locations that they occupy in the vascular tree from which they are isolated. Recently, DNA microarrays have indicated a significant variety in the gene expression pattern between endothelial cells from large vessels and microvessels [23]. Moreover,

15.2 Culturing Brain Capillary Endothelial Cells

Fig. 15.1 Isolation procedures of brain capillary endothelial cells. (A) General procedure to isolate endothelial cells from cerebral microvessels. (B) Isolation of bovine brain capillary endothelial cells (BBCECs), procedure from Meresse et al. [26]. (C) Isolation of mouse brain capillary endothelial cells (MBCECs), procedure from Coisne et al. [28].

Murugesan et al. [24] suggested that the induction of chemokines induced by lysophosphatidylcholine is different in microvascular endothelial cells compared to that of large vessel endothelial cells. Concerning brain vessels, Song and Pachter [25] showed a differential expression of BBB markers along isolated brain microvascular segments with obvious and progressive changes in the expression of these markers with the vessel size. Only vessels which can be considered as capillaries showed the BBB phenotype. Thus modeling the BBB requires a careful isolation and culture of primary endothelial cells coming from brain capillaries. Several procedures and many modifications have been used to obtain endothelial cell primary cultures. The species and source of cerebral microvessels vary from one laboratory to another. Commonly, the first steps of such procedures use mechanical and/or enzymatic means to disperse brain tissue in order to collect brain vessels (Fig. 15.1 A) [26–29]. However, enzymes should be used

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with care, as their actions are not well controlled and the endothelial cell cultures prepared in this way may be in fact a mixture of cells of capillary, arteriolar and venular origin. Percoll, albumin or sucrose gradients used after enzymatic digestion limit the nonendothelial cellular types, but do not prevent primary culture from being a heterogeneous mixture of endothelial cells. It is thus advisable to separate and discard the largest vessels from brain homogenate before using any enzymes for endothelial cell preparation (Fig. 15.1 B, C). Different and successive filtrations can be used for this purpose. Indeed, no obvious differences except size are detectable between venules, arterioles and capillaries. However, depending on the mesh porosity used, noncapillary vessels may be retained and the noncapillary origin of endothelial cell may still remain [30]. One way to reduce noncapillary vessels is to collect the smallest vessels from the brain homogenate, even if this may reduce the yield.

15.2.1.2 Endothelial Cell Culture From Capillaries Once the capillaries are isolated, different ways are described to obtain endothelial cells. In our laboratory, bovine brain capillary endothelial cells (BBCECs) have been obtained using an approach which does not use any enzymatic digestion (Fig. 15.1 B) [14]. After capillary isolation by mechanical homogenization and successive filtrations, microvessels consisting mainly of capillaries with a few tufts of arterioles and venules are seeded onto dishes coated with an extracellular matrix secreted by bovine corneal endothelial cells. Since only capillaries adhere to the extracellular matrix, arterioles and venules can easily be discarded. Five days after seeding, the first endothelial cells migrate out of the capillaries and start to form microcolonies. Endothelial cells are collected and amplified by successive trypsinizations (Fig. 15.2). Using this method, capillary endothelial cell origin and purity of the culture can be attested. However, the success of this nonenzymatic preparation depends mainly on the ability of endothelial cells to undergo cell division in tissue culture after trypsinization. For mouse brain capillary endothelial cell (MBCEC) preparation, the mechanical method described here was not sufficient. Indeed, endothelial cells did not migrate easily from seeded isolated mouse capillaries and enzymatic digestion of these vessels was required for MBCEC growth out. For these reasons the following modifications of the “BBCEC method” were made (Fig. 15.1 C): (1) the final selection of capillaries on bovine corneal endothelial cells was replaced by several filtration steps using 60-lm meshes to avoid any contamination with noncapillary vessels, (2) the capillary filtrate obtained was digested in collagenase/dispase, (3) after several washes, digested capillaries were seeded onto tissue culture-inserts. At 48 h after plating, MBCECs formed a confluent monolayer (Fig. 15.3) [28].


Fig. 15.2 Bovine brain capillary endothelial cell isolation procedure. Phase contrast micrographs of: (a) bovine cerebral capillaries seeded on a plastic dish coated with an extracellular matrix secreted by bovine corneal endothelial cells, (b) endothelial cells growing out from capillary and forming a microcolony, (c) confluent monolayer of bovine brain capillary endothelial cells: subculture from colony. Bars = 50 lm.

15.2.1.3 Primary Endothelial Cells and Subculture of Brain Capillary Endothelial Cells According to the literature and to our own experiences, the viability and proliferation of cerebral endothelial cells appear species-dependent (Table 15.1). Better outcomes are obtained when cells are obtained from bovine and pig species. This can explain the use of endothelial cell subcultures for in vitro BBB models in these two species. In our laboratory, bovine endothelial cells migrate from capillaries onto an extracellular matrix secreted by bovine corneal endothelial cells, as described before. When the colonies are sufficiently large, the largest islets are trypsinized and seeded onto 35-mm diameter gelatin-coated dishes. Endothelial cells from one 35-mm diameter dish are then harvested and seeded onto 60-mm diameter gelatin-coated dishes. After 68 days, confluent cells are subcultured at the split ratio of 1 : 20. Cells at the third passage are stored in liquid nitrogen. The subculture technique enables us to circumvent the limitations of primary cultures and to provide large quantities of these monolayers. Indeed, endothelial cells can be cultured from passage three after thawing to passage eight, corresponding to 48 generations.

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Fig. 15.3 Mouse brain capillary endothelial cell isolation procedure. Phase contrast micrographs of: (a) vascular components in mouse brain homogenates, (b) cerebral capillaries isolated from vascular components, (c) capillaries after collagenase/dispase digestion, (d, e) mouse brain capillary endothelial cells at 24 h (d) and 5 days (e) after seeding on matrigel-coated plastic dish. Bars = 50 lm. Micrographs were obtained from Coisne et al. [28].

Concerning mouse brain endothelial cells, viability and proliferation after trypsinization appear trickier and so far no real success in mouse endothelial cell subculture has been described. These cells show low rates of amplification, lack of development of real endothelium and loss of BBB phenotype. This fact explains why most attempts to model BBB model in rat and mouse use primary endothelial cells and why authors have used transfection to obtain immortalization [31]. Unfortunately, the cerebral phenotype can be modified in these conditions [32, 33].

15.2.1.4 Immortalization Immortalized endothelial cell lines have been established by introducing genes, such as the SV40 large T-antigen gene, into primary cultured cells. The first obvious problem is the potential loss of contact inhibition after immortalization which, as explained below, can result in an incomplete cerebral phenotype. Many authors circumvent this problem by using conditional cell division being able to stop growth. Temperature-sensitive SV40 large T-antigen is inactivated by shifting the culture temperature from 33 to 37 8C, under which condition cell


343

Table 15.1 Examples of in vitro BBB models developed in commonly used species. Abbreviations used: ACM astrocyte conditioned medium, c-GT c-glutamyl transpeptidase, mdr multidrug resistance, Pe studies permeability studies, SEM scanning electron microscopy, TEER transendothelial electrical resistance, TEM transmission electron microscopy, TJ: tight junctions. Species

Primary or subcultured endothelial cell

Isolation procedure

Endothelial cell

Coculture

BBB phenotype

Ref.

Cattle

Subcultured

Mechanical

From capillaries

With rat glial cells

ZO-1, occludin, P-gp, LDL and transferrin receptors, TEER, c-GT, drug transport screening, Pe studies, in vivo correlation

70

Cattle

Subcultured

Mechanical and enzymatic

From capillaries

ACM

TEER, ZO-1

13

Pig

Subcultured

Enzymatic

From microvessels

No

TEER, Pe studies

29

Human

Primary

Enzymatic

From microvessels

No

TEER, P-gp, Pe studies

71

Macaque

Subcultured


From capillaries

With macaque astrocytes

GLUT-1, c-GT, TJ in TEM, 69 VCAM-1/E-selectin (±TNF-a)

Rat

Subcultured

Enzymatic

From microvessels

With rat astrocytes

TEER, c-GT, OX-26, TJ in SEM

72

Mouse

Primary


From capillaries

With mouse glial cells

Occludin, claudins, JAM-A, Pe studies, TEER, Pg-P, CAMs (±LPS) MECA-32 negative, AHNAK

28

Mouse

Primary


From capillaries

No

Occludin, ZO-1

25

Mouse

Subcultured


From microvessels

No

GLUT-1, occludin, mdr, CAMs (±TNF-a)

39

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growth is repressed or arrested [34]. Unfortunately, even so, a loss of BBB properties is observed. Is it the transfection method? Is it the disruption of expression of essential genes by the transgene? Or is it the fact that, at the beginning, these cells do not have any brain capillary endothelial cell phenotype? So far, no explanation has been given. Recently, conditionally immortalized cells have been established by using transgenic mice [35] or rats [36] harboring the temperature-sensitive SV40 large T antigen. Using these transgenic animals, Hosoya et al. [37, 38] have established conditionally immortalized brain capillary endothelial cells (TM-BBB, TRBBB). As reviewed by Terasaki et al. [31], these two cell lines showed more BBB features than traditionally transfected cells. However, TR-BBB and TM-BBB do not form rigid tight junctions and may not be used for permeability studies. In conclusion, although immortalized cell lines possess obvious culture advantages, their use should be avoided for in vitro permeability studies. But, some of these cells do possess some in vivo functions, which, when determined, may be used for in vitro studies.

15.2.1.5 Purity The problem of contamination with nonendothelial cells has been encountered by anyone isolating brain capillary endothelial cells. Indeed, depending on the method used, fibroblasts, astrocytes and mostly pericytes have been observed as contaminating cells. The first two cell types are usually discarded when endothelial cells are isolated from brain microvessels. However, the separation of endothelial cells from pericytes, which are inherent to the capillary basal lamina, is trickier. The nonenzymatic method used for bovine brain capillary endothelial cell isolation in the laboratory allows the separation of endothelial cells and pericytes by microtrypsinization [14]. This separation step is even more important, as bovine endothelial cells will be subcultivated. Indeed, the presence of a few pericytes in the primary culture may lead to a real colonization of endothelial cells by pericytes after a few trypsinizations, as pericytes thrive under endothelial cell culture conditions. Different methods have been described to get rid of pericytes. Song and Pachter [25] and Wu et al. [39] described an endothelial cell purification step using magnetic beads. Perrière et al. [40] used the fact that P-glycoprotein expression is much higher in brain capillary endothelial cells than in contaminating cells, so that the cell-toxic P-glycoprotein substrates enter the contaminating cell and cause their death. Other authors, working with primary endothelial cell subcultures, utilize a brief trypsinization step as the endothelial cells approach confluence. Thus, endothelial cells are readily detached, while pericytes or smooth muscle cells still adhere to the substratum [30, 41]. In our mouse procedure [28], collagenase/dispase digestion of isolated capillaries promoted the migration of endothelial cells. Hoechst staining and tight junction immunolabeling showed that mouse brain capillary endothelial cells


Fig. 15.4 Immunocytochemical characterization of confluent mouse brain capillary endothelial cell monolayer cocultivated with mouse glial cells. (a) PECAM-1: whole cell surface presents dense distribution of immunoreactive dots. (b) Nuclear Hoechst staining and alpha-actin labeling of pericytes (arrow). (c–f) Cell-border localization of tight junction-associated proteins: (c) claudin-5, (d) occludin, (e) claudin-3, (f) JAM-A. Bars = 25 lm. Micrographs were obtained from Coisne et al. [28].

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formed a complete monolayer on matrigel-coated inserts (Fig. 15.4). The endothelial phenotype was confirmed by the expression of von Willebrand factor and PECAM-1 (Fig. 15.4 a). However, the digestion of capillaries could generate some contaminating pericytes (Fig. 15.4 b). FACS analysis showed that such cells were scarce compared to PECAM-1 positive cells (5% vs 95%). This low level of contamination was obtained without the use of any endothelial cell purification step and remained stable as experiments were made with primary endothelial cells. Furthermore, in spite of these contaminating cells, mouse brain capillary endothelial cells formed a well differentiated in vitro BBB (see below). Over-proliferation of pericytes can disturb the investigation of BBB properties, such as studies of brain capillary endothelium permeability. However, efficient methods have been developed and are now available to remove this cell type from in vitro endothelial cell culture. Paradoxically, in parallel with the development of these methods, more and more studies have revealed pericyte influences on cerebral endothelium [16]. The development of endothelial cell coculture with pericytes and study of pericyte influences on BBB properties should be attentively followed up, since the role of pericytes may have been underestimated until now [42].

15.2.1.6 Species As mentioned before, the success of in vitro BBB model establishment seems to be species-dependent. So far, no explanation has been given. However, two major hypotheses may be stated: (1) the longevity of each species may determine the capacity of the cells for in vitro cell division, (2) the culture conditions and reagents, e.g. the use of bovine serum, may currently be much better optimized for bovine than for murine species. If the availability of tools and technical difficulties may influence the choice of the species, the decision has to be made according to what the model will be used for. If the establishment of a well differentiated human in vitro BBB model is one major priority, other species such as bovine, murine, macaque (which have so far allowed progress in BBB understanding) are still valuable tools for future investigations. In our laboratory, the significance of developing an in vitro BBB model using murine tissue is supported by the fact that many in vivo models of chronic neuro-inflammation are developed in mouse and it also presents the possibility for creating a BBB model from gene-targeted (KO) animals, to complete in vivo studies.

15.3 Characteristics Required for a Useful In Vitro BBB Model

15.2.2 Coculture

In this chapter entitled “Modeling the Blood-Brain Barrier”, we must also mention the crucial role of brain capillary surrounding cells in the induction, and also in the maintenance and regulation of BBB properties. As mentioned above, numerous studies have shown the role of astrocytes, pericytes, microglia and recently, neurons [19, 43]. Recent data showed that a mixture of astrocytes and microglia was more efficient than purified astrocytes with regard to endothelium responsiveness, suggesting that intercommunication between each type of brain capillary cells may be important [15, 44]. Some studies also showed that endothelial cells are responsible for the astrocyte phenotype, emphasizing the reciprocal interrelations between coculture partners [45–48]. This fact should guide researchers in their choice of in vitro culture partners as it may determine the responsiveness of the system. In our laboratory, a coculture of bovine brain capillary endothelial cells and rat glial cells has been validated as an in vitro BBB model. Although useful, this heterologous model may lead to misinterpretations concerning coculture partner interrelations. For the study of inflammatory events, we have developed a syngenic in vitro model comprising of a coculture of mouse brain capillary endothelial cells with mouse glial cells. When considering endothelial cells, attention should be paid to the choice and use of endothelial cell partners, such as glial cells. Immortalized cell lines may not be as efficient as primary cells in endothelial BBB property induction [49].

15.3 Characteristics Required for a Useful In Vitro BBB Model 15.3.1 Confluent Monolayer

As mentioned above, the BBB is first an endothelium in which cells have to be jointly packed and in which cell contact inhibition is essential for monolayer function. Regulation of receptor expression is usually dependent on cell-cell contact and apposition. For example, LDL receptor expression and function are regulated by confluence. Indeed, we described a switch of this receptor from an endothelial cell lipid supplier during cell growth to a receptor allowing the transport of LDL after the cerebral endothelium was found (Fig. 15.5) [50]. Confluence of endothelial cells is also necessary to evaluate the endothelial permeability.

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Fig. 15.5 Effect of filipin (a cholesterol-binding agent causing the disassembly of caveolae) on the DiI-LDL endocytosis by bovine brain capillary endothelial cells. Comparison of filipin effects on BBB differentiated endothelial cells (A, B) and endothelial cells in growing phase (C, D). A vs B shows reverse effect of filipin: (A) DiI-LDL endocytosis after filipin treatment, (B) DiI-LDL endocytosis after filipin removal. Bar = 50 lm. C, D: DiI-LDL endocytosis was carried out with brain capillary endothelial cells without (C) or with (D) pretreatment with filipin. Micrographs were obtained from Dehouck et al. [50].


15.3.2 Tight Junctions and Paracellular Permeability

The presence of well differentiated tight junctions is one of the principal characteristics of the BBB, as these structures reduce paracellular transport, which can be considered as nonspecific transport through the cerebral endothelium. In this way, tight junctions participate actively in the selective barrier that contributes to the maintenance of brain homeostasis. In another way, just as confluence is important for receptor expression and function, tight junctions are essential for the regulation of endothelial cell polarity, contributing to protein distribution at the cell surface and distinguishing the luminal and abluminal faces of the endothelium [51, 52]. In vitro cerebral endothelium polarity is particularly important to evaluate bidirectional and selective exchanges between brain and blood. For these reasons, the presence and efficiency of tight junctions must be evaluated before an in vitro endothelium is chosen as a BBB model. Tight junctions are complex structures made of different proteins, such as occludin, claudin-5, claudin-3 and JAM-A (see Chapter 4) [53, 54]. To test the presence of tight junctions in vitro, immunostaining of these proteins can be assessed (Fig. 15.4). Caution must be taken, as the presence of several of these proteins on the cerebral endothelium is still in debate. This is the case for claudin-1 and claudin-3. In vivo, the claudins found in cerebral endothelial cells were first claudin-5 and claudin-1. Discrepancies concerning the presence of either claudin-1 or claudin-3 on cerebral endothelium were due to the use of a cross-reactive antibody [55]. The presence of occludin, claudin-5, claudin-3 and JAM-A on in vitro endothelium is a first step to study tight junctions; and the peripheral distribution of these proteins is one step further, because they were described as tight junctional transmembrane molecules believed to restrict permeability [54]. However, studies have shown that peripheral expression of these proteins is not sufficient and does not always correlate with the tightness of these structures [55, 56]. Indeed, as the role and regulation of tight junctions is not yet completely understood, their immunolocalization has to be completed with other studies, such as the evaluation of endothelium permeability for low molecular weight and hydrophilic molecules which do not interact actively with endothelial cells. The attenuated flux of these molecules (e.g. sucrose, inulin, Lucifer yellow, HRP) emphasizes the presence and efficiency of tight junctions on in vitro monolayers. Furthermore, the presence and efficiency of tight junctions reduce or control the exchange of charged molecules through the BBB. Transendothelial electrical resistance (TEER) directly corresponds to the “tightness” of in vitro endothelium. In our laboratory, this assessment is used during the establishment of the BBB barrier in vitro model. However, for recent experiments, permeability studies have replaced TEER as a measurement tool, as in our hands they are more sensitive, more reproducible and can easily be performed in the different laboratories working on this topic. We should mention that coculture of endothelial cells with glial cells is essential for a good in vitro tight junction differentiation and that the in vitro presence

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of well differentiated tight junctions is important, not only to complete our knowledge of their structures and regulation but also to go further into BBB property investigation. Indeed, not only paracellular transport but also vesicularand receptor-mediated transport depends on the expression of these tight junctions [14, 57].

15.3.3 Transcellular Transport, Receptor Mediated Transport

For reasons discussed in the former paragraph, in vitro transcellular transport studies should be performed on endothelia presenting well differentiated tight junctions. Numerous receptors have been described as participating in nutrient transport through the BBB [58, 59]. The presence and function of these receptors can be checked to validate in vitro BBB models. In vitro expression of these proteins can either be used to study transcytosis of nutrients (Fig. 15.5) [50, 60], the transcytosis signalling pathway and/or to investigate cerebral drug delivery [61–63]. The next paragraphs list a few examples of receptor/protein expression depending on endothelium barrier function. As with tight junctions, most of the studies focus on astrocyte influence on receptor expression and vesicular transports. Recently, Berezowski et al. [16] have shown that pericytes also alter BBB features. As mentioned before, P-glycoprotein (P-gp) is an important characteristic of BBB but is often lost in culture conditions, so the presence of this protein attests to the congruence of the model with the in vivo situation (Fig. 15.6). P-gp is a flippase involved in certain drug transport, thus its presence is a crucial issue when using in vitro models to study BBB drug transports. The functionality of the P-gp can be shown using the transport of vincristine, a P-gp substrate [64]. Also, multidrug resistance-associated proteins have been recently located in brain capillary endothelial cells and their expression is regulated by either astrocytes or pericytes [16]. Expression of these emerging proteins should be considered to study BBB drug transport (see Chapter 19). Other proteins with unknown functions have been investigated in the BBB. The lack of MECA-32, a mouse antigen commonly described on nondifferentiated brain endothelial cells and noncerebral endothelial cells in vivo, suggests a good differentiation of the in vitro cerebral endothelium [65]. Recently, Gentil et al. [66] identify AHNAK as a protein marker of endothelial cells with barrier properties. Present in vitro, this protein relocates from the cytosol to the plasma membrane when endothelial cells acquire BBB properties (Fig. 15.6).


Fig. 15.6 (a, b): Characterisation of confluent mouse brain capillary endothelial cell monolayer cocultivated with glial cells: immunofluorescence of (a) P-gp (mAb: C219), (b) AHNAK, (c, d) AHNAK immunolabeling on confluent bovine brain capillary endothelial cell in “solo” culture (c) or cocultured with rat glial cells for 12 days (d) [66]. Bars = 25 lm.

15.3.4 Expression of Endothelial Adhesion Molecules/Vascular Inflammatory Markers

Cerebral endothelium disturbances observed in CNS infections emphasize the necessity for in vitro BBB models that mimic immune events. One approach to validate the relevance of a BBB model for studying conditions of inflammation is to examine the expression of adhesion molecules/vascular inflammatory markers on the in vitro endothelium. BBB responsiveness to inflammatory treatments can also be tested, focusing on endothelium permeability changes or the regulation of endothelial adhesion molecules. These in vitro studies should correspond to the literature regarding in vivo brain microvascular endothelium pathogenesis. Coisne et al. [28] have shown that LPS treatment of cocultures led to the upregulation of endothelial ICAM-1 and VCAM-1; and they referred to publications demonstrating the upregulation of ICAM-1 and VCAM-1 on cerebral endothelium in experimental autoimmune encephalomyelitis (EAE) [67], systemic lupus erythematosus (SLE) [68] and AIDS [69]. Furthermore, the establishment of an in vitro model of pathological BBB can be set up by either inducing a pathological environment [15] or isolating brain capillary endothelial cells from experimental disease-induced animal models or

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gene-targeted (KO) animals. These models will be valuable tools to complete the in vivo studies.

15.4 Conclusion

In vitro models are most useful when they closely mimic diverse in vivo situations. According to the literature, the establishment of a BBB model requires: (1) the provision of endothelial cells from cerebral capillaries, (2) the use of primary endothelial cells, (3) the coculture of these endothelial cells with glial cells. These three conditions are the most accurate ones, as they increase the success in obtaining a well differentiated endothelium which can be used as a BBB model. Because in vitro BBB have drawbacks and advantages, in vitro models are useful only if researchers are aware of the model limits. In any case, the use of in vitro models requires precautions; and checking the hypotheses in vivo still remains the most accurate proof. Under these conditions, modeling the BBB is possible and can be extremely valuable when correctly investigated with care.

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16 Induction of Blood-Brain Barrier Properties in Cultured Endothelial Cells Alla Zozulya, Christian Weidenfeller, and Hans-Joachim Galla

16.1 Introduction

Cerebral endothelial cells build up the inner microvascular wall, separating the blood from the brain parenchyma. These cells form a functional barrier limiting free exchange between the blood and the brain tissue proper. This structure is commonly known as the blood-brain barrier (BBB). The BBB provides homeostasis to the brain interstitium, and is essential for optimal neuronal functioning through the network of tight cell-cell contacts (tight junctions) between capillary endothelial cells. Therefore, the morphological substrate of the BBB is attributed to the specialized endothelial cells that strictly control the transcellular pathway of toxic and xenobiotic substances from the blood to the interstitial fluid of the brain [1]. The interendothelial tight junctions are specialized connections (see Chapter 4) that prevent the intercellular leakage of blood-borne substances into the brain parenchyma [2]. Tight junctions seal the interendothelial cleft; and as a consequence no paracellular shunt of drugs or solutes diffusion from the blood site into the CNS emerges. Due to these structural and morphological features, drug delivery to the brain is challenged (see Part V). To allow a better design of new pharmaceuticals and noninvasive strategies to delivery drugs to the brain tissue, a better understanding of the mechanisms which regulate the formation of the BBB is important. Although the endothelium is the principle barrier and communicating interface, the local microenvironment modulated by associated cells, such as astrocytes and pericytes, contributes to BBB function. Brain microvascular endothelial cells depend on constant support from both astrocytes and pericytes in order to maintain their BBB ability. The associated cells are in close contact with endothelial cells separated by basement membranes (BM) [3], which cover the abluminal surface of the endothelium. The BM is formed by extracellular matrix (ECM) proteins which are involved in the regulation of specific endothelial functions. The molecular components of ECM derive from the surrounding glia, pericytes, macrophages and endothelial cells, and seem to play an important Blood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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role in barrier formation. However, the molecular mechanism of ECM regulation at the BBB is not yet well understood. Cell cultures are an excellent tool to investigate molecular mechanism that underlie the formation of the BBB. The successful production of a BBB in vitro is strongly dependent on an adequate mimicking of the in vivo situation. In our laboratory, we established a primary culture of pig microvascular endothelial cells and very recently a second in vitro model based on a serum-free mouse endothelial culture system [4]. The pig system, with its high transendothelial electrical resistance (TEER) values up to 2000 X cm–2 and low permeability of about 2 ´ 10–7 cm s for sucrose, allows us to investigate transport processes to quantify the access of pharmacological substrates across the BBB into the brain. The newly developed mouse BBB model facilitates the application of molecular biological strategies currently available due to access to the mouse genome data. This allows in silico studies to design molecular tools like small interfering RNA, and antisense oligonucleotides, as well as knockout mice to elucidate the role of specific genes and proteins in BBB function. The application of the glucocorticoid hormone hydrocortisone (HC) in chemically defined serum-free medium was found to improve the BBB properties of both pig and mouse microvascular endothelial cell cultures [4, 5], leading to high values in TEER and low sucrose permeability. Obviously, the junction tightness is drastically improved under the influence of HC. This is important to note since such high barrier properties as described in our system have not been achieved by astrocyte/endothelial cell coculture systems so far. Moreover, we found that pig cerebral endothelial cells exhibit a reduced activity of matrix metalloproteinases (MMPs) when substituted with physiological concentrations of HC [6]. These proteinases (see Chapter 14) were found to be secreted by diverse cell types and it was also shown that MMPs degrade the major macromolecules of the brain ECM [7–9]. Serum was found to contain MMP-2 and MMP-9 molecules. Due to the high MMP activity in serum, the disruption of cell-cell contacts and proteolysis of the tight junction protein occludin was to be expected within in vitro systems [10]. Since MMPs balance the proteolysis, synthesis and assembly of the extracellular matrix, it became obvious that these compounds are involved in the differentiation of the BBB phenotype. New evidence will be described in this chapter indicating that the formation of cell-cell and cell-substrate contacts are reinforced by the endogenously produced extracellular matrix proteins derived from glial cells such as astrocytes and from pericytes. In essence, here we give a report, in conjunction with Chapter 15, on the establishment of in vitro BBB systems with the focus on pig and mouse in vitro models as well as on the involvement of ECM and MMPs to build up barrier properties. The described models can be used to study neurological disorders, inflammatory reactions of the CNS and the recruitment and transmigration of immune cells in diseases related to multiple sclerosis and human immunodeficiency virus (HIV)-associated syndromes.

16.2 In Vitro BBB Models

16.2 In Vitro BBB Models

The unique features of cerebral microvascular endothelial cells, as compared to peripheral blood vessels, are the presence of tight intercellular junctions, the paucity of pinocytotic activity [11, 12], high number of mitochondria [13], the absence of fenestrations [14] and the expression of specific marker enzymes like transferrin receptors [15] and c-glutamyl transpeptidase [16]. Among the variety of specialized properties, the most important feature is an efficient endothelial barrier with a low paracellular permeability, which can be easily probed by monitoring the passage of small solutes (for instance, sucrose or inulin) across the cellular monolayer. However, tracer methods have often limited time resolution. Therefore, tight junction complexity and barrier function are most sensitively and readily quantified by measuring the TEER [17]. Further progress in this field was made possible by the development of a new technique which allows the monitoring of cell-cell and cell-substrate contacts in vitro. This method has been coined electric cell-substrate impedance sensing (ECIS) [18]. In 1978, Panula and coworkers demonstrated that isolated rat brain microvascular endothelial cells can be cultured under in vitro conditions [19]. Today, the preferential sources of endothelial cells [20] derive from rat [21, 22], dog [23], cow [24–28], pig [29, 30] and primates [31]. Properly cultured cells exhibit typical features of microvascular endothelial cells, which include attenuated pinocytosis, lack of fenestrations and tight junctions [24, 32, 33], in addition to a typical protein expression pattern. However, higher reproducibility, more homogeneity, ease of propagation and a short population-doubling time were the reasons to develop cell lines derived from rat [34–39], cow [40, 41], pig [42] and human brain microvessels [43]. Nevertheless, due to the lack of barrier properties, none of these immortalized cell lines express all the features necessary for reliably modelling a BBB phenotype. Attempts to induce BBB differentiation in endothelial cell lines [36] have been performed, but most of them failed. Some cerebral endothelial cell lines have been shown to restrict the passage of macromolecules and have been used in studies of paracellular permeability. To overcome this problem, considerable efforts have been made to reinduce BBB properties in endothelial cells by coculturing them with astrocytes or glioma cells [28, 44–47], or by applying brain-derived differentiation factors. These studies indicate that the establishment and maintenance of the unique cerebral endothelial phenotype results from the neural milieu, in particular the specific interactions provided by astrocytes which surround the brain microvessels [48–50]. Whether physical cell-cell contacts are necessary for the induction of the BBB properties is not clear yet. Also, the use of conditioned media obtained from cultures of rat brain astrocytes or C6 glioma cells reveal some BBB-supporting capacity [28, 46, 51–56]. However, neither cocultures with astrocytes, nor addition of conditioned media induce the entire complement of BBB features found in vivo. Therefore, a better knowledge and appropriate technologies are essential to define new in vitro models for a better understanding of the molecular and biochemical features of the normal BBB.

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16.3 Hydrocortisone Reinforces the Barrier Properties of Primary Cultured Cerebral Endothelial Cells 16.3.1 In Vitro Model Based on Pig Brain Capillary Endothelial Cells (PBCEC)

An in vitro system consisting of pig brain endothelial cells allowed us to obtain cell cultures with TEER between 1000 X cm–2 and 2000 X cm–2 [57]. The paracellular permeability of 14C-sucrose indicating values as low as 2 ´ 10–7 cm s underlines the close proximity of this model to the situation found in vivo [58]. The expression of tight junction proteins was confirmed by Northern blot analysis and immunocytochemistry [59, 60]. Besides the barrier function indicated by high TEER values, typical efflux transport systems were found, such as the Pglycoprotein and the ABC-G2 transporter, a member of the multidrug resistance family [61]. Thus, this technology offers a model of fully differentiated brain endothelial cells and its applicability to studies on the physiology, pharmacology and pathology of the BBB [30, 62]. As described in Chapter 5, the integrity and BBB properties of the cerebral endothelium are enhanced by coculture of endothelial cells and astrocytes in filter setups [26], using astrocytes-conditioned medium (ACM) [28], or primary

Fig. 16.1 Serum withdrawal and addition of HC both improve the TEER of pig brain capillary endothelial cells (PBCEC). In the presence of serum, cells reach only low electrical resistances. Serum withdrawal allows the development of barrier properties with resistances of 500 X cm–2 and higher. HC was found to substantially reinforce the BBB properties of cultured PBCEC, leading to an increase of TEER up to 1200 X cm–2 [30]. Standard deviation is expressed for n = 10.

16.3 Hydrocortisone Reinforces the Barrier Properties

cultured cerebral endothelial cells in either astrocytes coculture and/or under ACM conditions [63]. Our in vitro system is based on the supplementation of culture medium with physiological concentrations of the glucocorticoid hormone HC. Previously, we have shown that withdrawal of serum leads to improved BBB properties [30, 62, 64] closely resembling the in vivo BBB phenotype. Figure 16.1 shows the effect of serum withdrawal and addition of HC on the barrier properties represented by TEER.

16.3.2 In Vitro Model Based on Mouse Brain Capillary Endothelial Cells (MBCEC)

As mentioned above, our pig-based in vitro BBB system reveals high transendothelial resistance when the cells are cultured under serum-free conditions in HC-supplemented medium [30]. The question arose whether this effect is species-specific or whether HC also exerts an effect on other primary cultures of brain derived microvascular endothelial cells. A newly established mouse model of the BBB exhibiting almost no contaminating cells in the form of astrocytes and pericytes [4, 65] was analyzed with respect to its response upon HC exposure. Cells in a confluent monolayer showed the typical spindle-shaped endothelial cell morphology and expressed both common and BBB-specific endothelial cell markers. Von Willebrand factor (factor VIII-related antigen) [66] as well as tight junction proteins such as claudin 5, occludin and ZO-1 were demonstrated to be expressed in MBCEC by immunofluorescence staining, Western blot, and quantitative real-time RT-PCR. Additionally, mRNAs of all tested BBB markers like c-glutamyltranspeptidase [16], alkaline phosphatase [67] and endothelial cell markers like angiotensin-converting enzyme [68] and transferrin receptor [15] were detected. A special property of the microvascular endothelial cells in the brain is the presence of numerous efflux transporters which protect the brain against toxic compounds. The expression of P-glycoprotein and ABC-G2 were found at mRNA levels, indicating that the mouse-derived brain endothelial cells express some main BBB-related transport proteins [4]. However, the measured transendothelial electrical resistance for MBCEC was much lower compared to cultured PBCEC in vitro. Hence, the TEER of PBCEC under serum-free conditions increased in the presence of HC, up to values of 1200–2000 X cm–2 (Fig. 16.1), depending on the cell culture conditions [69]. The MBCEC developed TEER values of only 200 X cm–2 in serum-free medium supplemented with HC, whereas serum-containing medium lowered the resistance of these cells down to 50 X cm–2 (Fig. 16.2). The effect was time-dependent and detectable after a lag period of 2 h (data not shown). In contrast to the observed increase of the paracellular resistance in the pig system, the withdrawal of serum induced a TEER decrease to about 30 X cm–2 for mouse endothelial cells (Fig. 16.2). These data indicate that the primary culture of MBCEC expressed all the typical markers of cerebral endothelial cells, but lacked the strong barrier

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Fig. 16.2 The combination of serum withdrawal with the addition of HC improves the transendothelial electrical resistance (TEER) of mouse brain capillary endothelial cells (MBCEC). Cells cultured with HC show a 5-fold higher TER than cells cultured in serum-containing medium, while the TEER of cells cultured without both supplements reveal the lowest values of resistance, compared to supplemented conditions. Standard deviation is expressed for n = 5.

properties. This lack of tightness may be the consequence of an incomplete tight junction formation in culture caused by a dedifferentiation in response to the in vitro situation. However, the measured low values of transendothelial resistance in MBCE were in the range of other reported cell culture systems [5, 54, 70]. Additionally, differentiation of MBCEC in vitro has been described to be different from the in vivo situation [70, 71].

16.4 The Involvement of Serum Effects

Cell cultures require a number of different factors that are normally supplied by the added serum. It is well known that fibronectin, growth factors, hormones and extracellular matrix proteins are common components of serum-containing media which can influence cell proliferation and differentiation. It is thus rendered difficult to unveil cell biological aspects of differentiation in the presence of serum. As mentioned before, the withdrawal of serum significantly reinforces the BBB properties of pig cerebral endothelial cells (Fig. 16.1). The inhibition of serum factors favoring the formation of tight cell-cell contacts was recently described by us [60]. This finding is supported by other groups which corroborate the ability of serum to alter both the permeability and electrical resistance of epithelial and endothelial monolayers. As reported by Chang et al. [72], serum induces a breakdown in the junction tightness of cultured retinal-pigmented epithe-

16.5 Hydrocortisone Improves the Culture Substrate

lial cells. Alexander et al. [73] have further shown that lysophosphatidic acid (LPA) decreases the permeability for cyanocobalamine in bovine pulmonary aortic endothelial cells (BPAEC). English et al. [74] looked for an effect of LPA on TEER and observed an increased value of electrical resistances in BPAEC treated with LPA [74]. In contrast to this, Schulze et al. [75] noticed that LPA under physiological conditions decreases TEER, and increases the permeability in pig cultured endothelial cells [75]. Vascular endothelial growth factor (VEGF), a secreted highly endothelial-specific mitogen [76] and a potent angiogenic factor [77] (see Chapter 2), is also known to influence the permeability and electrical resistances of vascular endothelial cells [78–80]. Additionally, previous experiments have shown that VEGF causes an increase in the albumin permeability of cultured cow retinal microvascular endothelial cells [81], increases retinal vascular permeability of fluorescein in vivo [82], stimulates an increase in water permeability in cow aortic endothelial cell cultures and induces an increase in paracellular permeability of human umbilical vein endothelial cells [83]. Further data obtained by us revealed that both VEGF and LPA decrease TEER in PBCEC in vitro [60]. We identified several compounds of serum which are able to influence TEER in vitro [60]. Among them was a 67-kDa fraction which could be detected by MALDI and which was proven to significantly decrease the PBCEC TEER in vitro. We also observed that heat-inactivated serum induces a decrease in TEER similar to nonheat-inactivated serum, although to a minor extent. At that time, the 67-kDa factor found by Nitz et al. [60] in serum was not completely analyzed. However, this molecule was further identified as matrix metalloproteinase type 2 [6]. When discussing the effect of serum on cultured brain microvascular endothelial cells, it should be taken into account that sera from different species evoke different effects. Fetal calf serum and ox serum cause a stronger effect on PBCEC as compared to pig serum. Horse and human sera have almost no effect on PBCEC. These data indicate that species differences do exist, a fact that must be considered when PBCECs are exploited.

16.5 Hydrocortisone Improves the Culture Substrate by Suppressing the Expression of Matrix Metalloproteinases In Vitro 16.5.1 ECIS Analysis of Improved Endothelial Cell-Cell and Cell-Substrate Contacts in HC-Supplemented Medium

As mentioned above, the electrical resistance provides a measure of junction tightness and endothelial monolayer integrity. Generally, establishment of TEER is considered without taking into account cell-cell and cell-substrate interactions. This fact may lead to false conclusions with respect to the mechanism of barrier formation, since some mediators seem to influence TEER by acting on the cellmatrix interaction and/or the cell-cell contacts. The application of a newly devel-

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Fig. 16.3 Schematic representation of the model of Giaever and Keese [89] to calculate cell topology, including the formation of cell-cell (Rb) and cellsubstrate (a) interactions, as well as the capacitance of the cell membrane (Cm). These parameters allow a description of the barrier and adhesion properties of cells.

oped ECIS technique allows us not only to measure the dynamics of cell adhesion, spreading and tight junction formation on electrode surfaces, but also to discriminate between different morphological parameters of the cell monolayer [18, 84–87]. The parameters for cell-substrate contact (a) and cell-cell contact (Rb; Fig. 16.3) were defined as essential values which determine TEER. A change in Rb detected by ECIS indicates an alteration in paracellular resistance, while increasing values for a indicate a closer cell-substrate contact. We applied this technique to our BBB in vitro model to assess the effects of HC and serum

Fig. 16.4 Results of ECIS readings. The parameters a and Rb are presented as weighted average values for n = 5. HC addition increases parameter a compared to serum-treated cells, indicating that the formation of cellsubstrate interaction is influenced by the glucocorticoid hormone. Likewise, Rb values point to a strengthening of cell-cell contacts induced by HC.

16.5 Hydrocortisone Improves the Culture Substrate

on the formation of cell-cell and cell-substrate contacts. Endothelial cells closely attached to the substrate inhibit the flow of ions underneath. An increase in extracellular matrix proteins reduces the electrical current beneath the cells. In cell cultures with low electrical resistances, like mouse cerebral endothelial cells, an increase in a becomes more important and contributes more to overall TEER. These data clearly indicate that both a and Rb have to be taken into account when effects on TEER are considered. Figure 16.4 shows that higher values for both a and Rb can be detected in HC-containing medium. This observation shows that the paracellular electrical permeability of MBCEC is reduced mainly by Rb, which provides evidence for improved cell-cell contacts. The increase in a after HC treatment may be multifactorial and we will discuss this in the following.

16.5.2 Low Degradation of ECM in HC-Supplemented Medium Leads to Improved Cell-Substrate Contacts of Cerebral Endothelial Cells

It is well known that glucocorticoids increase the barrier properties of vascular cells [88–90]. It has also been shown that HC increases barrier properties in terms of TEER and permeability for 14C-mannitol in epithelial cells [91]. A similar effect was found in a breast cancer tumor cell line [92]. Glucocorticoids are able to influence the expression of MMPs. These enzymes play an important role in the remodeling of the extracellular matrix proteins [7–9] by proteolytic degradation (see Chapter 14). There is evidence that the expression of MMPs can be down-regulated by medium supplemented with glucocorticoids [93]. Our recent studies provide evidence that PBCECs are involved in the secretion of MMPs at the BBB. HC was shown to be able to suppress endothelial MMP secretion, resulting in increased barrier properties [6]. This finding strongly suggests a role for MMPs in the regulation of BBB permeability. In accordance with this result, we presume that a from ECIS readings can be increased by optimizing the integrity of ECM under HC treatment. Higher endothelial MMP secretion in HC-free cultures would lead to a consecutive degradation of ECM proteins, resulting in fewer cell-substrate contacts and consequently a lower a value. Immunocytochemical analysis showed that MMP molecules remain firmly attached to the extracellular matrix substrate when cells are depleted by lysis. We also observed a significant decrease in the amount of MMPs after incubation of endothelial cells in HC-supplemented medium. Furthermore, we discovered that increased endothelial MMP activity at the BBB leads to MMP-dependent cleavage of the tight junction protein-occluding and a profound disruption of cell-cell contacts [10]. This could explain the lower Rb value derived from ECIS readings. The described effects can be taken as evidence that HC improves both the cell-cell (tight junctions) and the cellsubstrate interaction by partly inhibiting MMP activity at the BBB.

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To prove the ability of HC to modify cell-substrate contacts by suppressing the expression of MMPs, we investigated the basolateral MMP secretion by endothelial cells, using immunostaining. We found that MMPs remain bound to the extracellular matrix of endothelial cells when secreted. Thus, the endogenous ECM derived from first generation of endothelial cells grown in HC-free medium are likely to be different from ECM derived from cells cultured in HCsupplemented medium. We then studied the influence of modified endogenously produced ECM by secreted MMPs on the formation of cell-cell and cell-substrate contacts, using ECIS readings. The ECIS data were recorded as a function of time (unit: hours) and are depicted in Fig. 16.5. The capacitance recorded at 40 kHz was used as a measure for endothelial cell-substrate contacts. The cells attach to the substrate with an almost spherical morphology (Fig. 16.5 A, C). Therefore, the area of contact between the basement membrane and the cell surface is remarkably small. It increases continuously while the cells spread out. Due to the increase in cell attachment and spreading, the capacitance continuously drops down. Thus, the dynamics of the reduction in capacitance could be read as the process of increasing adhesion, which leads to further attachment and spreading of endothelial cells on the electrode surface. The resistance of the system at 400 Hz was taken as a measure for endothelial cell-cell contacts. Cultured cells with highly developed tight junctions exhibit elevated electrical resistance values (Fig. 16.5 B, D). Details of this experiment have been described elsewhere [18]. To investigate the influence of HC on ECM expression, BCEC were first cultured in serum-free medium with or without HC. The time-course of capacitance and resistance of PBCEC measured by ECIS under these conditions is shown in Fig. 16.5 A, B. The cells became completely confluent in less than 10 h after plating and they adhered in the same manner under both culture conditions, with and without HC. As mentioned before, HC improves the barrier properties of brain endothelial cells and thus increases the transendothelial electrical resistance (Fig. 16.5 B). After 30 h of ECIS measurements, the cells were lysed, leaving the ECM attached to the electrode surfaces. By doing this, two different endogenously derived types of ECM were coated on the electrode surfaces: the plus-HC ECM and the minus-HC ECM. A second generation of primary cultured cerebral endothelial cells in serum-free medium was then seeded onto the endogenously derived ECM of both plus-HC and minus-HC conditions. ECIS readings of the capacitance and resistance over time for the second PBCEC generations in serum-free medium without HC supplementation are shown in Fig. 16.5 C, D, respectively. In the case of the endogenously derived ECM from endothelial cells cultured without HC supplementation, there is a delay in the time-course of cell attachment and spreading compared to PBCEC seeded on the ECM derived from the cells cultured before exposure to HC (Fig. 16.5 C). A plausible explanation is that MMP molecules secreted by the first generation of endothelial cells remained present on the ECM of cells cultured in HC-free medium. These MMPs apparently modified the matrix proteins in a way that the second generation of BCEC could not attach and spread

16.5 Hydrocortisone Improves the Culture Substrate (A)

(B)

(C)

(D)

Fig. 16.5 Electric cell-substrate impedance sensing (ECIS) readings of capacitance over time (A, C; measured at 40 kHz) and resistance (B, D; measured at 400 Hz) for bovine cerebral endothial cultures (BCEC). The cells were directly cultured on gold ECIS electrodes in serum-free medium with (solid lines) and without (broken lines) hydrocortisone (HC) at 550 nM. BCEC cultured in the presence of HC developed higher electrical resistances than cells grown in HC-free medium. A second generation of endothelial

cells was cultured in serum-free medium without HC on the extracellular matrix (ECM) material derived from the first endothelial cell generation cultured with (solid lines) and without (broken lines) HC. The time-course of capacitance (C) indicates a time-shift in the adhesion of these cells to the substrate. The resistance (D) also indicates lower values for endothelial cells grown on the ECM material derived from the cells cultured in HC-free medium. (Units: capacitance in nF, resistance in X).

as fast on the ECM generated by endothelial cells cultured in HC-supplemented medium. The time-shift detected for the endothelial cells in the form of a delayed attachment phase on minus-HC ECM coating could be explained by the increased time needed by the second generation of cells to produce their own extracellular matrix components. Thus, HC was again shown to suppress the degradation of a proper extracellular matrix, probably by an inhibited secretion of MMPs. This is also indicated by the electrical resistance (Fig. 16.5 D). The second generation of endothelial cells grown on matrix material derived from

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the first generation of cells grown in HC-supplemented medium developed a higher resistance in a shorter time (Fig. 16.5 D).

16.6 The Role of Endogenously Derived ECM for the BBB Properties of Cerebral Endothelial Cells In Vitro

As described, astrocytic endfeet are in close proximity with capillary endothelial cells (see Chapter 9) and are separated from the vessels only by pericytes and the BM. The close proximity of astrocytic endfeet to brain capillaries attracts continuous interest and has led to the idea that astrocytes convey a modulator function to BBB properties [63, 94]. Pericytes comprise a further class of cells found in close association with endothelial cells (see Chapter 5). They may be involved in the regulation of vascular permeability and transport processes across the BBB [1]. Further studies have indicated that signals provided by the interactions between capillary endothelial cells and the surrounding neuroglia, including the ECM, induce the BBB phenotype [95]. By measuring the electrical properties of cerebral endothelial cells cultured on filters covered with different purified extracellular matrix proteins, it was suggested that the BM placed between the endothelium and surrounding astrocytes may be involved in the differentiation of the BBB phenotype [96]. The molecular mechanisms through which both ECM and glial cells affect the BBB are still poorly understood. To get a better insight into this interaction we aimed to investigate the role of ECM proteins endogenously derived from astrocytes and pericytes. For these purposes, barrier formation by BCEC cultured on astrocyte- and pericyte-derived ECM was studied by ECIS and compared to resistance values obtained on ECMs produced by cerebral and aorta endothelial cells. Aorta endothelial cells lack barrier properties and are leaky. The extracellular matrix derived from these cells was used as a control substrate, representing a type of ECM differing from that derived from brain endothelial cells. The rationale for this experimental design derived from observations which showed significant differences in the barrier development of BCEC on endogenously produced ECM as compared to purified individual ECM molecules [97]. The resulting resistance values obtained in these experiments are summarized in Fig. 16.6. Note that the resistances given here are measured at 400 Hz and should be not confused with normally reported (X cm–2) resistance values. The functional significance of purified ECM molecules for the development of electrical resistances increases from laminin (5 ± 1 ´ 103 X) to fibronectin (19 ± 2.5 ´ 103 X), with fibronectin being the best exogenous substrate for the promotion of cell-cell interaction. Although the PBCEC cultured on fibronectin acquired high resistance, endothelial cells grown on cellular basement membranes derived from precultured tight BCEC developed an even better barrier. When PBCEC were cultured on ECM derived from pericytes, PBCECs developed an electrical resistance of 25 ± 2 ´ 103 X. On an astrocytic extracellular matrix, BCEC developed more than

16.6 The Role of Endogenously Derived ECM for the BBB Properties of Cerebral Endothelial Cells

Fig. 16.6 The influence of purified individual ECM molecules (laminin, collagen type I + III, collagen type IV, fibronectin) and endogenously derived ECM from brain cells (endothelial, pericytes, astrocytes) compared to nonbrain ECM (aorta ECM) on the transendothelial electrical resistance of BCEC monolayers. The cells were cultured in serum-free medium supplied with HC. The values for electrical resistance measured at 400 Hz are depicted after 55 h of culturing the BCEC. Data given are means ± SD for one out of two ECIS assays, all of which were conducted in duplicate.

33 ± 2.5 ´ 103 X. Interestingly, the endogenously produced ECM derived from aorta endothelial cells only slightly improved the tightness of PBCEC (cell-cell contacts). Although the lowest electrical resistances for endogenously derived ECM was found in aortic ECM (10 ± 2.5 ´ 103 X), these values were still significantly higher than that assessed for the cells cultured on laminin coating (5 ± 1 ´ 103 X). A feasible explanation of this finding is that the complexity of endogenous ECM can not be mimicked by a single-component coating of ECM substrates. From all endogenously derived substrates, the extracellular matrix derived from astrocytes was the best ECM for PBCEC in terms of its attachment and capacity to form tight junctions. With respect to the methodological approach, ECIS is a useful tool in measuring the potentials of endogenously derived ECMs on brain endothelial cells. The production of some common integrins and ECM molecules in astrocyte-endothelial cell cocultures has been already described by Wagner et al. [98]. They found that astrocytes secrete laminin-5, which is bound to the basement membrane of cerebral microvessels by its integrin receptor a6b1. A physical contact possibly involving the binding of endothelial a6b1 integrin receptors and laminin-5 was found to be responsible for the induction of astrocytic laminin [98]. Based on our ECIS experiments, it is tempting to speculate that astrocytic lami-

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nin-5, among other ECM molecules, may reside on the electrode surface after the lysis of astrocytes during ECM denudation. Since PBCEC were found to secrete an elevated level of a6b1 receptor as found by flow cytometry (own unpublished observations), the formation of high resistance in PBCEC monolayers on astrocyte-derived ECM can be explained by the secretion of integrin receptors on endothelial and/or astrocytic cell surfaces and their interactions with matrix proteins. Additionally, we found that astrocytes express inhibitors for MMPs and exhibit low expression levels of MMP-2 and MMP-9 enzymes. Schiera et al. [99] previously reported that cocultures of brain endothelial cells with astrocytes and neurons leads to the stabilization of tight junctions and the formation of a functional BBB. We suggest that MMPs released by astrocytes as well as by pericytes may activate growth factors bound to the ECM, which then exert their influence through specific receptor complements on the differentiation and growth of BCECs. Obviously, a complex interplay of a variety of components including suitable adhesion molecules and their complementary receptors, as well as matrix proteinases in conjunction with their specific inhibitors, are orchestrated in a way that leads to a BCEC phenotype which mimics a suitable BBB culture model.

16.7 Conclusions

Two serum-free BBB models derived from pig and mouse brains are described for the investigation of the BBB function. These cells depend on serum in the growth medium to proliferate. Serum, however, inhibits the formation of cellcell contacts, as demonstrated by the decrease in the transendothelial electrical resistances in monolayers. Withdrawal of serum from monolayers significantly improves the barrier properties of pig capillary endothelial cells, but not of cells obtained from mouse brains. Both pig and mouse brain endothelial cultures express higher TEER values when incubated in serum-free medium and in the presence of hydrocortisone [4, 30]. Besides serum factors, the BM conveys some important function to the BBB, since its degradation by MMPs leads to a disruption of the BBB integrity. Improved BBB properties observed under the influence of HC hints at the ability of this hormone to suppress the secretion of MMPs. The level of secretion of these enzymes was found to correlate with the integrity of the BBB in the form of its electrical resistance [10]. Extracellular matrix proteins secreted by the endothelial cells in HC-supplemented medium seem to be less degraded, thereby improving the cell-substrate contacts of cerebral endothelial cells. The importance of the ECM synthesized by glial cells is also discussed. The extracellular matrixes derived from both astrocytes and pericytes were found to be most efficient with respect to the development of an endothelial barrier. The ECM components seem to contribute considerably to the development of the BBB properties of endothelial cells in vitro [97].

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C. M. Lo, C. R. Keese, I. Giaever 1995, Biophys. J. 69, 2800. P. Mitra, C. R. Keese, I. Giaever 1991, Biotechniques 11, 504. F. D. Ingraham, D. D. Matson, R. L. McLaurin 1952, N. Engl. J. Med. 15, 568. J. O. Jarden, V. Dhawan, J. R. Moeller, S. C. Strother, D. A. Rottenberg 1989, Ann. Neurol. 25, 239. K. Yamada, Y. Ushio, T. Hayakawa, N. Arita, N. Yamada, H. Mogami 1983, J. Neurosurg. 59, 612. K. S. Zettl, M. D. Sjaastad, P. M. Riskin, G. Parry, T. E. Machen, G. L. Firestone 1992, Proc. Natl Acad. Sci. USA 89, 9069. P. Buse, P. L. Woo, D. B. Alexander, H. H. Cha, A. Reza, N. D. Sirota, G. L. Firestone 1995, J. Biol. Chem. 270, 6505. C. Jonat, H. J. Rahmsdorf, K. K. Park, A. C. Cato, S. Gebel, H. Ponta, P. Herrlich 1990, Cell 62, 1189. I. Isobe, T. Watanabe, T. Yotsuyanagi, N. Hazemoto, K. Yamagata, T. Ueki, K. Nakanishi, K. Asai, T. Kato 1996, Neurochem. Int. 28, 523. S. Fischer, M. Wobben, J. Kleinstuck, D. Renz, W. Schaper 2000, Am. J. Physiol. Cell Physiol. 279, C935. T. Tilling, D. Korte, D. Hoheisel, H. J. Galla 1998, J. Neurochem. 71, 1151. A. Zozulya 2004, Westfälische Wilhelms-Universität, Münster. S. Wagner, H. Gardner 2000, Neurosci. Lett. 284, 105. G. Schiera, E. Bono, M. P. Raffa, A. Gallo, G. L. Pitarresi, I. Di Liegro, G. Savettieri 2003, J. Cell Mol. Med. 7, 165.

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17 Artificial Blood-Brain Barriers Luca Cucullo, Emily Oby, Kerri Hallene, Barbara Aumayr, Ed Rapp, and Damir Janigro

17.1 Introduction

The current view of the BBB has shifted from a purely anatomic concept to a more physiological and dynamic vision. This change was brought about by evidence that the BBB actively regulates the transport of substances from blood to brain and provides active shielding to many potentially noxious xenobiotic substances. Initial studies of the BBB were performed in vivo, mostly to determine the permeability of compounds across the brain endothelium. This approach offered valuable information about the behavior of different classes of compounds and helped identify specific BBB transport systems (e.g., injection single-pass method to measure cerebrovascular transport and permeability introduced by Oldendorf in 1970). However, further characterization of the BBB, especially at the cellular and molecular level, was delayed due to the complex environmental conditions present in vivo. These complexities stimulated the development of in vitro experimental approaches, such as new cell culture techniques and improved technologies to monitor BBB function (see Chapters 15 and 16). Thanks to these advancements, the BBB has been characterized in detail both physiologically and morphologically [1–6]. The BBB is found in the brain of all vertebrates; and in humans it is formed in the first trimester of fetal life [7]. Morphologically, the BBB is formed by specialized ECs lining the intraluminal portion of brain microvessels together with the closely associated astrocytic endfeet processes sharing the basal lamina and enveloping more than 98% of the BBB endothelium [8]. Astrocyte interaction with the cerebral endothelium determines BBB function, regulates protein expression, modulates endothelium differentiation and appears to be critical for the induction and maintenance of the tight junctions and BBB properties [9–14]. The microvascular endothelium at the BBB level is characterized by the presence of tight junctions (zonulae occludentes), a lack of fenestrations and minimal pinocytotic vesicles. BBB EC characteristics include:


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· · · · · · ·

few pinocytic vesicles, tight junctions with small hydrophilic pores (15–20 Å), fractional area: 0.01%, high negative charge density at the surface (sulfated glycoproteins), low permeability to water soluble molecules (electrolytes), 15% (by weight) of BBB cell membranes is lipid (other cells: 50%), P-glycoprotein efflux system.

These distinct morphological properties of the BBB account for the “restraining” nature of brain capillary ECs. In particular, tight junctions between the cerebral ECs form a diffusion barrier (Fig. 17.1) which selectively excludes most bloodborne substances and xenobiotics from entering the brain, protecting it from systemic influences. However, the BBB does not act as an absolute barrier, since non-polar molecules (lipid-soluble substances such as alcohol, narcotics, anticonvulsants) pass with ease. The transit across the BBB involves translocation through the capillary endothelium by carrier-mediated transport systems. The BBB also provides specialized transport systems for nutrients and other biologically important substances (i.e., d-glucose, lactate, phenylamine, choline, adenosine, arginine, adenine) from the peripheral circulation to neurons in the parenchyma. The transport systems at the BBB include: 1. Uptake at the BBB · hexoses (e.g., glucose), · amino acids, · peptides, · acetylated low density lipoprotein (scavenger receptor), · transferrin (transferrin receptor), · organic cations, · monocarboxylic acids, · nucleosides. 2. Efflux at the BBB · neurotransmitter metabolites, · uremic toxins, · organic anions, · immunoglobulin c (immunoglobulin fc receptor), · P-glycoprotein, · multidrug resistance proteins. Because of its selectivity, the BBB plays a crucial role in the determination of neurotoxicity and its prevention by specific transport mechanisms [15, 16]. Unfortunately, some of these efflux transport systems that developed to protect the brain from potentially dangerous substances may also contribute to the phenomenon known as multiple drug resistance (MDR) during the treatment of several CNS disorders, such as drug refractory epilepsy or intractable brain tumors [17–19]. The necessity to develop alternative pharmaceutical strategies that bypass the shielding of brain parenchyma and to study new therapeutic

17.1 Introduction

approaches for many neurological diseases demanded the rapid development of cell culture-based in vitro models that are able to reproduce the physiological, anatomical and functional characteristics of the BBB. These cell culture systems have the potential to be valuable tools in unraveling the complex molecular interactions underlying and regulating the permeability of the cerebral endothelium in normal and pathological conditions, as well as allowing for transendothelial permeability screening and a better prediction of drug penetration across the BBB. This explains why the aim of in vitro models is to functionally express as many unique characteristics of the BBB cerebral endothelium in vivo as possible. The understanding of the BBB physiology is critical for complex issues such as pathogenesis of neurological diseases involving BBB dysfunction (e.g., brain tumors, ischemia, hypoxia, brain edema, multiple sclerosis, meningitis) and the development of pharmaceuticals that can cross the BBB. In fact, CNS drug design (e.g., antineoplastics, antivirals, antiepileptics) cannot rely entirely upon physico-chemical properties to cross the BBB. For example, lipophilicity alone is a poor predictor for drug penetration into the CNS since it relies on a passive, diffusional type of uptake. Many lipophylic drugs are potential substrates for efflux carriers of the BBB (particularly P-glycoprotein) that can drastically reduce their penetration into the brain [20]. In the following paragraphs, we provide an overview of the characteristics of cell culture-based models of the BBB available today and insights into ongoing development of future devices.

Fig. 17.1 Brain microvasculature. Mouse brain was perfused with FITCalbumin. Note the lack of fluid extravasations due to the BBB.

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17.2 Requirements for a Good BBB Model

There are significant quantitative and qualitative differences between the various BBB cell culture systems, but there are specific requirements common to all that need to be addressed in order for the model to be as functional and accurate as possible. The ingredients of a good in vitro BBB model include: · expression of tight junctions between ECs; · low permeation to sucrose or electric current flow; · selective and asymmetric permeability to physiologically relevant ions, such as Na+ and K+; · selective permeability to molecules, based on their oil/water partition coefficient and molecular weight; · expression of drug-metabolizing enzymes; · functional expression of mechanisms of active extrusion such as multidrug resistance proteins; · cell-cell interaction and relative exposure to as yet unidentified “permissive” or “promoting” factors (presumably secreted by astrocytes); · exposure of the apical membrane to shear stress to promote growth inhibition and differentiation of ECs; · similarity in responsiveness to permeation modulators; · ease of culture and low cost. The expression of tight junctions between ECs (determining a restrictive paracellular permeability) and low permeation to polar molecules that do not have a specific carrier-mediated transport system (e.g., sucrose) are mandatory conditions. Moreover, a functional BBB model must guarantee a selective permeability to molecules, based on their oil/water partition coefficient and molecular weight, and selective and asymmetric permeability to physiologically relevant ions, such as Na+ and K+. Furthermore, in order to obtain a functional BBB, the model should allow for the development and maintenance of mechanisms of active extrusion such as multidrug resistance proteins (e.g., Mdr1, Pgp) naturally present in vivo and for the expression of “drug” metabolizing enzymes. Cell-cell interactions leading to physiologically more natural structural design and relative exposure to “permissive” or “promoting” factors, mostly released by surrounding glia, are also required. Also, in order to be an effective research tool, the in vitro BBB should be easy to culture, able to assure data reproducibility and be as inexpensive as possible. Finally, a new wave of research has demonstrated that circulating blood cells are critical elements for the establishment of a functional in vitro BBB environment, capable of reproducing CNS-impairment-related phenomenons (e.g., inflammation, stroke, brain trauma, etc.) that occur in vivo.

17.4 Cell Culture-Based In Vitro BBB Models

17.3 Immobilized Artificial Membranes

Immobilized artificial membrane (IAM) chromatography is a technique used for the analysis and purification of many biological molecules. IAM has been proposed as a model for predicting drug permeability across the blood-brain barrier [21]. Originally developed by Charles Pidgeon at Purdue University, the IAM stationary phase consists of a monolayer of phospholipid covalently immobilized on an inert silica support. The resulting IAM surface is a chemically stable chromatographic material which simulates the exterior of a biological cell membrane. IAM chromatography can be used for the analytical and preparative separation of membrane-associated proteins and for the non-covalent immobilization of membrane associated proteins. IAM chromatography has gained acceptance for the chromatographic estimation of the membrane permeability of small-molecule drugs [22, 23]. In particular, IAM chromatography can be used to estimate drug permeability and to measure phospholipophilicity. However, this system has several limitations: (a) the IAM does not mimic the dynamics of fluid membranes, in particular lateral diffusion, (b) it is impossible to simulate diffusion across a membrane bilayer, (c) the IAM can model only drug permeation through cells where equilibrium across the membrane is the rate-limiting step, and (d) the IAM cannot mimic active extrusion.


In the early 1990s, in vitro models of the BBB started to emerge which had the potential to be valuable tools in unraveling the complex molecular interactions underlying and regulating the permeability of the cerebral endothelium in normal and pathological conditions and allowing for transendothelial permeability screening. These in vitro methods are based on isolated brain capillaries or the isolation and subsequent culture of brain capillary ECs in static, and more recently, in flow-based (dynamic) in vitro apparati. Other approaches implicate the use of immortalized cell lines (e.g., RBE4, MDCK) [24, 25]. All these models attempt to mimic the complexity of the mammalian BBB, but each of them is characterized by a different selective permeability to different compounds and may manifest a different range of trans-endothelial electrical resistance (TEER) values. TEER is a measure of the electric (ionic) conductance of the monolayer and is a useful measure of the “tightness” of the monolayer. Before entering into a detailed description of the cell culture-based models of the BBB, we want to provide a general overview of the cell types currently used to establish an in vitro BBB.

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17.4.1 Cell Lines

Several cell lines have been used as biological substrates for BBB modeling. Usually, one favors cell lines that are easier to expand instead of relying on more time-consuming primary culture of brain ECs.

17.4.1.1 Immortalized Rat Brain Endothelial Cells The immortalized rat brain endothelial (RBE4) cell line is probably the most extensively characterized cell line used for BBB modeling. Unfortunately, no data are available on how RBE4 behave when cultured under dynamic conditions, since all data available were obtained under static conditions. These ECs have been transfected with a plasmid containing the E1A adenovirus gene. The RBE4 has been shown to functionally express a number of BBB transporters and mechanisms of active extrusion (e.g., Pgp, Mrp1) [26–32]. An immortalized cell line has the advantage that cell cultures can be expanded at will, which drastically reduces cost and labor. However, some disadvantages of using an immortalized cell line include the fact that the cells have been genetically manipulated and generally form incomplete tight junctions, thus leading to the establishment of a less functional and less selective BBB. This drawback is evident when looking at permeability data for a known blood volume marker molecule, such as sucrose. These data indicate that cell lines form very “leaky” monolayers and lack the necessary paracellular barrier properties to be considered a BBB permeability screen [33]. One possible explanation may be that ease of expansion is obtained by molecular strategies that alter cell cycle checkpoints. Desai et al. [34] have shown that reduced proliferation is a determinant of endothelium differentiation perhaps allowing for astrocytic influences to “tighten” the BBB.

17.4.1.2 Other Cells From Non-Cerebral Sources Mardin-Darby canine kidney (MDCK) cell lines are widely used in the establishment of an in vitro BBB. BBB models based on MDCK cell monolayers show low permeability to sucrose, and because of the ease with which these cells can be grown; they are used for the screening of passively transported CNS compounds. The MDCK cell line is comprised of different clones with different properties. The MDCK-I clone achieves the lowest permeability to sucrose but there are also MDCK cells available that are transfected with the human MDR-1 gene (MDCKmdr-1 cell line) which lead to a polarized overexpression of P-glycoprotein [35]. However, these non-cerebral cells differ from brain ECs (BCEC) with respect to morphology, transport properties, metabolism and growth (e.g., lack of contact-induced inhibition of the cell proliferation mechanism that is present in brain ECs). Morphological differences compared to cerebral ECs are likely to be reflected in drug-cell membrane interactions resulting in not only different transport-mediated permeation but also transfer mediated by passive


diffusion [36, 37]. The use of intestine derived cell line Caco-2 in the establishment of an in vitro BBB has also been taken into consideration [38]. However, differences in drug permeability between the intestine and brain-derived cells are significant, thus limiting the use of Caco-2 cells [39].

17.4.1.3 Brain Capillary Endothelial Cell Cultures Highly purified populations of cultured microvascular cells for the study of the developmental and pathophysiological processes of the BBB became available in the early 1980s. Brain microvessel ECs can be isolated by mechanical dispersion (homogenization, filtration, sieving, centrifugation), enzymatic procedures using collagenase or a combination of both mechanical dispersion and enzymatic digestion from brain microvessels in culture [40–42]. With these techniques, a viable and homogeneous population of brain capillary ECs can be obtained for the establishment of a BBB culture system. Primary or low-passage brain capillary cells generally provide the closest phenotypic resemblance to the in vivo BBB phenotype such as bovine brain ECs. The immediate availability of bovine brain tissue in combination with the relative ease with which pericyte free clones can be obtained and subcultured have led to the extensive use of bovine brain as a source for ECs. Bovine brain ECs are commonly used to establish BBB models as monoculture or co-cultured with astrocytes. Another convenient source of brain ECs are derived from pig brain (PBEC) and yield a sufficient number of brain ECs to allow extensive permeability testing. Rat brain ECs have also been utilized but have the main disadvantage of low yield of capillary fragments (due to the relatively small brain) and a tendency for a negative impact of pericytes and other contaminants on the permeability of the monolayer, as compared to cells from other tissue sources. Furthermore, while a number of drug resistance molecules are expressed in rodent brain, the human brain appears to be much more complicated as evidenced by both clinical (e.g., multiple drug resistance in epileptics) [43] and in vitro studies (expression of various drug resistance molecules in different tissues, expression of brain tumor markers in known brain tumors, epileptic brain, etc.) [44]. Given the complexity and specificity of the interactions at the BBB level, the use of primary cultures of human brain ECs (in coculture with human astrocytes) rather than cell lines can provide important insights (e.g., improved accuracy of drug permeability values due to maintained substrate/transporter specificity) in the study of drug refractory brain disease and in the prescreening and optimization of new drug formulations. However, the availability and ease of obtaining human specimens in order to isolate cells can be a challenging obstacle; and the large number of cells to be isolated is crucial to run a successful experiment. Thus, for industrial purposes, human cells are still an unrealistic alternative. Although these pitfalls seem major, the benefits of using primary human cultures far outweigh the costs. Human specimens provide research opportunities for a variety of etiologies that otherwise may be impossible to recapitulate in cell lines or even primary culture from rodent, pig or cattle CNS. The establishment of such

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humanized in vitro models can provide accurate representations of many neurological diseases involving BBB dysfunction in drug refractory patients.

17.4.2 Monoculture-Based In Vitro BBB Models

Cell culture systems have been developed to reproduce the key properties of the intact BBB and to allow for testing of the mechanisms of transendothelial drug permeation. The most common and easy apparatus to culture EC is the Transwell system. This apparatus was developed based on a simplified view of the BBB. The Transwell system consists of a porous membrane support (polycarbonate or polyester) submerged in culture medium. This model is characterized by a side-by-side vertical diffusion system, where cultures of ECs derived from various sources are grown on semi-permeable membranes to allow for nutrient exchange (Fig. 17.2 A, B). Attractive features of this model (Table 17.1) are its simplicity (easy to culture with minimal skill requirements) and the ability to perform multiple experiments at the same time (one type of drug per well, or different concentrations of the same drug in each well). Furthermore, this model is ideal for Michaelis-Menten kinetics of transport due to fixed volumes in each compartment and is very cost-efficient [45]. However, BBB properties in vivo (e.g., expression of tight junctions) are bestowed on ECs by the surrounding physiological environment (e.g., shear stress, astrocytes, pericytes, blood cells) [46–48]. In the absence of these physiological stimuli, the ECs lose their “BBB properties”, limiting this model to results with little predictive value (Fig. 17.2 C). For example, it has been shown that ECs grown in monoculture under static conditions may lack the expression of specific transporters and tight junctions, leading to abnormal permeability across the EC layer [49]. Thus, the sucrose permeability (Psucrose) of these monolayers ranged from 10–4 cm s–1 to 10–5 cm s–1, compared with 10–6 cm s–1 to 10–8 cm s–1 in vivo [5]. As a consequence of an elevated Psucrose, BBB permeability values of other compounds can also be overestimated (Fig. 17.2 C, D). The TEER in vivo is estimated to be ca. 1500 X cm–2, whereas the measurements in endothelial monolayers cultured in vitro vary over 20–200 X cm–2. However, it has been demonstrated that the addition of hydrocortisone or dexamethasone to the culture media seems to provide significant BBB “tightening” effects even in the absence of glia, thus improving barrier function [50, 51]. Endothelial cells at the BBB level in vivo present a functional specialization of the luminal and abluminal membranes [52]. This polarity was initially demonstrated by Betz and Goldstein in 1978 and is reflected by the preferential expression and distribution of membrane transporters and enzymes that act to protect the CNS and to promote substrate delivery to the brain parenchyma maintaining brain homeostasis [53–55]. Another factor to be considered is the fact that in vivo only the luminal side of ECs is exposed to serum proteins, while the ab-

17.4 Cell Culture-Based In Vitro BBB Models Table 17.1 Advantages and disadvantages of current BBB models.

Pros

Mono-dimensional

Bi-dimensional (coculture)

Tri-dimensional flow-based (DIV-BBB)

Ease of use

Coculture allows induction of BBB properties

Artificial capillaries connected by gas-permeable tubing allows source of growth medium, exchange of O2 + CO2, exposure to flow

Ideal for MichaelisMenten kinetics of transport due to fixed volumes in each compartment

Molecule flux across is lower than monodimensional

Induction of BBB properties

Can do several experiments at same time

Cost-efficient

High TEER

Cells are easier to isolate for further study

Low permeability to sucrose

Minimum skills

Stereoselective transport

Cost-efficient

Can mimic I.V./I.A. vs P.O. delivery Long-term studies possible Presence of drug extrusion mechanisms

Cons

Lacks physiological flow

Lacks physiological flow

Linear kinetic studies more difficult, due to flow

High permeability to sucrose

High permeability to sucrose

Cost

Very low TEER

Low TEER (to a lesser extent)

Skills

Exposure to serum on both sides (luminal + abluminal)

Number of cells to be loaded/ used

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Fig. 17.2 In vivo versus in vitro monoculture-based BBB: the Transwell apparatus. (A) Shows a typical Transwell apparatus, while (B) is a schematic representation of a Transwell diffusion chamber. The microporous membrane allows for the free passage of nutrients and diffusible factors between the luminal and abluminal compartments. (C) Lists the permeability values of clinically relevant compounds both

in vivo and in vitro (Transwell). Note how the permeability values obtained in a Transwell apparatus (D) are often misleading (significantly higher). (E) Is a schematic representation of in vivo BBB versus a monoculture-based in vitro BBB. Note that in vivo, only the luminal side of ECs is exposed to serum proteins, while the abluminal one is exposed to perivascular influence (e.g., astrocytes).


luminal part is either exposed to glial influence, basal lamina or cerebrospinal fluid (Fig. 17.2 E). ECs grown as a mono-dimensional layer upon a porous membrane are exposed to serum, on both the luminal (intravascular) and the abluminal (parenchymal) side. This non-physiological condition may accelerate the dedifferentiating process that the ECs experience and further enhance the loss of the BBB characteristics with serial cell passage. Furthermore, an increased cell cycle rate due to lack of antimitotic influences by laminin and flow will cause ECs to pile up in a multilayer fashion [56, 57]. EC grown in static monoculture-based models generally have a short lifespan compared to flow-based culture models. In addition, the occurrence of irregular patterns of cell adhesion or “edge effect” in Transwells can seriously hamper the measurement of BBB permeability.

17.4.3 Coculture-Based In Vitro BBB Models

Although primary cultures of brain endothelium alone may form tight intercellular junctions, coculture with astrocytes results in the increased formation and complexity of endothelial tight junctions and expression of specific BBB markers [58], including the physiologic glucose transporter isotype (GLUT-1), P-glycoprotein, GGTP and OX-26 [5]. The presence of glia and the establishment of glial-endothelial interactions has also been shown to increase the expression of brain endothelial marker enzymes (e.g., c-GTP, alkaline phosphatase, acetylcholinesterase, Na+/K+-ATPase), MDR proteins (e.g., P-glycoprotein) and tight junctions and to help create a phenotype more closely resembling that found in vivo. A recent study performed in rat brain capillaries has provided evidence that functional expression of a typical ATP-binding cassette, G2 (rat homologues rABCG2) on the luminal side of brain capillaries is up-regulated by astrocyte-derived soluble factor(s) concomitantly with the induction of its specific mRNA [59]. Coculture with astrocytes also increase barrier tightness, as shown by higher TEER values compared to monoculture models. This increase is even higher when dexamethasone or l-a-glycero-phospho-d-mio-inositol (GPI) is added to the culture medium [60]. By the use of the Transwell apparatus previously described, the coculture can be established to enable cell-cell contact via astrocytic endfeet (astrocytes and ECs are seeded on opposite sides of the porous support) or without any contact by seeding the astrocytes at the bottom of the well and the ECs on the porous support. The coculture model is useful for studying the functionality of the BBB as well as transport-related processes and interactions between ECs and glia [61]. The major advantage of the bi-dimensional model compared to the mono-dimensional one is the establishment of conditions more closely resembling the brain microanatomy (Table 17.1). Finally, recent studies have shown that the use of conditionally immortalized brain capillary EC lines (derived from transgenic animals harboring temperature-sensitive SV40 large Tantigen gene) in coculture with pericytes and/or astrocyte cell lines, can provide

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a BBB model capable of retaining the in vivo transport rate of several compounds and various forms of gene expression [62]. However, coculture models of BBB still lack the presence of shear stress (intraluminal blood flow), which has been demonstrated to further differentiate the ECs and to play a central role in the cerebrovascular system by promoting the differentiation and maintenance of the BBB phenotype [5, 6, 10, 34, 56, 63–69].

17.5 Shear Stress and Cell Differentiation

Endothelial cells in vivo are continuously exposed to shear stress, a tangential force generated by the flow of blood across their apical surfaces. Shear stress affects EC structure and function, such as cell orientation with flow direction, distribution of cell fibers, induction/suppression of genes [70–72], production of vasoactive substances and improved cell adhesion [73–75] and mitotic arrest in ECs [34, 57, 76] and is also able to induce metabolic changes [34, 64]. Turbulent, but not laminar shear stress stimulates EC turnover [77]. An acute increase in shear stress induces increased release of prostacyclin and nitric oxide [78, 79] and a decrease in endothelin-1 mRNA [80]. Microvascular ECs cultured under flow are larger in volume and show an abundance of microfilaments, endocytic vesicles and clathrin-coated pits [74]. In addition, steady laminar shear stress inhibits DNA synthesis of ECs. The inhibition of the cell transition from the G0/ G1 to the S phase of the cell cycle is due to up-regulation of the cdk inhibitor p21 [81]. Accordingly, in vivo the DNA synthesis of EC preferentially occurs at branch orifices, with low flow rates, for example where atherosclerotic processes are initiated. High laminar shear stress promotes glycosaminoglycan synthesis, tight junction formation and the expression of junction-related proteins [82, 83]. Thus, it is clear that biomechanical forces generated by blood flow play a role in the induction of many BBB properties. Interestingly, EC are not the only cells responsive to shear stress. All cells in the circulation respond to physiological shear stress, including erythrocytes, platelets and leukocytes [84–86]. Given the considerations summarized above, it is not surprising that attempts were made to culture ECs under the influence of shearing forces. An EC monolayer (even from non-brain origin) can be exposed to a quasi-laminar or pulsatile shear stress by the use of a purpose-built cone and plate viscometer. These systems can be modified to accept culture plates of different sizes. Cells are seeded on the bottom of the plate and the shear stress is generated by a rotating cone which transmits the shear force to the cells through the culture media (Fig. 17.3). The angular velocity and the cone angle determine the level of shear stress generated. Pulsing and reversing the angular velocity of the cone can be used to achieve backflow or pulsatile shear stress.

17.6 Flow-Based In Vitro BBB Systems

Fig. 17.3 Schematic representation of a cone and plate viscometer. An EC monolayer can be exposed to a quasi-laminar or pulsatile shear stress by the use of a purpose-built cone and plate viscometer. Note that l is viscosity, m is kinematic viscosity, x is angular velocity and a is cone angle. The angular velocity and cone angle determine the level of shear stress generated.

17.6 Flow-Based In Vitro BBB Systems 17.6.1 Dynamic In Vitro BBB: Standard Model

The dynamic in vitro model of BBB (DIV-BBB) originates from a modification of a cell culture system used for hybridoma cell expansion. This in vitro “cell differentiation factory” provides quasi-physiological experimental conditions for culturing ECs and astrocytes in a capillary-like structure and is able to functionally and anatomically mimic the brain microvasculature. The DIV-BBB is characterized by a pronectin-coated microporous polypropylene hollow fiber structure that enables coculturing of EC (intraluminally) with glia (abluminally). The entire system is connected to a media reservoir via gas permeable silicon tubes that allow for the exchange of O2 and CO2. A servo-controlled variable-speed pulsatile pump generates flow from the media source through the capillary bundle and back. One-way valves positioned on either side of the “pump” ensure unidirectional flow (Fig. 17.4 A). The pumping mechanism is capable of generating flow levels of 1– 50 ml min–1 with associated shear stress levels of 1–200 lN cm–2. Shear stress levels are estimated to be in the range of 1–5 lN cm–2 at the capillary level in vivo but the pump allows changes in shear stress levels at any time point without changing oxygen tension and/or glucose levels. The DIV-BBB also enables realtime continuous monitoring of BBB function by measurement of TEER across the barrier via electrodes inserted in the luminal and abluminal (ecs) compartments. Exposure to

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Fig. 17.4 Flow-based in vitro BBB (DIV-BBB). (A) Shows a schematic representation of a typical DIV-BBB. Endothelial cells are seeded intraluminally in fibronectin-coated hollow fibers, while astrocytes are cultured extraluminally. (B) Shows waveforms of pressure changes in pre- and post-DIV-BBB capillary segments

mimicking the physiological changes in vivo. (C) Shows a cross-sectional view of artificial capillaries employed in our model. Note that ECs grow in a multilayer fashion in the absence of luminal flow (right side). When flow is present (left side), ECs grow in a typical monolayer comparable to what observed in brain microvasculature.

controlled pumping rates makes the model suited for the study of EC responses to levels of shear stress corresponding to large arteries (ca. 70 lN cm–2). Note, however, that as implemented today, the system does not allow studying the effects of turbulent or otherwise altered flow. The pulsatile flow generated consists of a complex waveform with a substantial drop of pressure occurring at the end of the capillaries, giving these models the ability to reproduce the hemodynamic conditions observed in vivo (Fig. 17.4 B). Under these conditions, ECs develop a morphology that closely resembles the endothelial phenotype in situ [73], demonstrating that ECs grown with flow develop greater differentiation than after static culture (Fig. 17.4 C). This improved cellular differentiation is also reflected by much higher TEER


Fig. 17.5 BBB permeability in vivo vs in vitro. (A) Shows the permeability of clinically relevant compounds in vivo and in a DIV-BBB. Note how the permeability values obtained in a DIV-BBB (B) are comparable to permeability in vivo. (C) Shows typical TEER values measured in vivo, in the DIV-BBB and in Transwells. Note how the DIV-BBB model closely compares to the in vivo situation.

(> 1200 X cm–2) and drug permeability values that reproduce the in vivo scenario (Fig. 17.5). Typical BBB properties in ECs grown in hollow fibers under dynamic condition include low permeability to intraluminal potassium, negligible extravasation of proteins and the expression of a glucose transporter. In addition, culturing ECs with glia affects the overall morphology of the cells and induces the expression of BBB-specific ion channels [56, 64, 87, 88]. However, this model has limited applicability due to its design. The compartmentalization of

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ECs and glia in the hollow fiber system in the presence of pulsatile flow makes the study of linear kinetics more complex. The cylindrical shape of the apparatus does not allow for visualization of either compartment to assess morphologic and/or phenotypic changes of the cells of interest. Additionally, the cell inoculation volume and media requirements are enormous, considering the fact that in general this model is not reusable. Finally, the volume and physical access to the ECs allow only for introduction of cell suspensions and not tissue slice preparations. In spite of these limitations, several reports have described the usefulness of the DIV-BBB for molecular [89], pharmaceutical [65, 90–93], morphological and functional studies [10, 34, 56, 64, 66, 67, 94, 95].

17.6.2 Dynamic In Vitro BBB: New Model

The new DIV-BBB (NDIV-BBB), recently developed, maintains all the dynamic characteristics that are attractive in the standard DIV-BBB but also includes novel features unavailable under the previous configuration (see Fig. 17.6 panels A and B for a side-by-side comparison and a detailed technical description). The apparatus consists of a rectangular polycarbonate hollow chamber enclosed by a permanent glass bottom and a removable clear acrylic top. The chamber contains an adjustable number of artificial polypropylene capillaries. These capillaries have an inner diameter of 600 lm, a wall thickness of 200 lm and 0.64-lm trans-capillary pores that allow free diffusion of solutes from the abluminal to the luminal compartment and vice versa. Several improvements in the design of the dynamic BBB model allow for longitudinal studies of the effects of flow and coculture in a controlled and fully recyclable environment that simultaneously permits visual inspection of the abluminal compartment and manipulation of individual capillaries. These modifications have expanded capabilities for research involving the central nervous system and neurological diseases. This model in particular has several advantages compared to the previous one: 1. The ability to expose and remove single or multiple capillaries during the course of an experiment for cell isolation (luminal and/or abluminal), morphological and/or phenotypic studies [56]. 2. The possibility of visualizing cell-cell interactions with inverted light microscopy as well as techniques involving fluorescence imaging. 3. The number of cells required to establish a functional BBB is one-fourth of the standard model. 4. It is not limited to EC and astrocyte coculture, since the model allows for inclusion of other relevant cell types (e.g., neurons) that can be seeded on the bottom of the abluminal chamber. 5. The model can be customized to accommodate different numbers of capillaries.


Fig. 17.6 Cross-comparison between DIV-BBB model technical specifications.

However, this model is not designed for industrial use where numerous tests have to be quickly performed on a large scale (e.g., pharmaceutical companies). Furthermore, the system setup requires greater technical skills and more time than any other model previously described, limiting the use of this apparatus.

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17.7 A Look Into The Future: Automated Flow Based In Vitro BBBs

The need for novel pharmaceutical strategies as well as the necessity to limit the increasing costs of experimental studies, pre-screening and testing is continuously pushing researchers to develop improved in vitro models to accelerate drug design. For this reason in the near future we will undoubtedly see DIVBBBs modified to provide higher predictability while being fully scalable, customizable and well suited for extensive or industrial use. New biotechnology companies are taking on the challenge (e.g., Flocel Inc.) by developing new models that can implement several features to make them ideal for large-scale usage as required by pharmaceutical companies. For example, the Flocel’s cartridges are built with more constrained dimensions in order to minimize the number of cells necessary to set up the system as well as to reduce the operating costs. This model features online computer-controlled TEER monitoring in sweep mode (all frequencies) or at fixed frequencies for a more detailed BBB integrity assessment. The electrodes are built into the cartridge to reduce the risk of external contamination (Fig. 17.7). A microdialysis probe (customized for different needs with different cut-offs) can be inserted into the capillary bundles and attached to a fraction collector which is also computer-controlled and fully programmable. Then, analysis can be conducted with the glucose/lactate analyzer, HPLC or other devices, depending on the customer’s criteria and purpose. Other apparati are being designed around the DIV-BBB, such as an external “turret-shaped” micro-incubator system that can be used to maintain the cartridges at the desired temperature, avoiding the problem of manipulating the cartridges in restrained spaces (classic incubator) and to automatically control/ adjust O2 and CO2 levels in the culture medium. The system is fully automated, thus minimizing operator efforts. Furthermore, several DIV-BBBs can be run at the same time (stackable units), yet remaining independent of one another. This is extremely important when different experimental paradigms need to be tested. Further advancement in this field will come with the introduction of new materials or more sophisticated techniques for the manufacture of capillary-like hollow fibers, where anchoring molecules for cell adhesion can be implemented in their matrix construction and thus allow for better physiological properties than those currently available today.

17.8 Conclusion

In this article we have provided a short review and analysis of different in vitro BBB models, from static side-by-side diffusion systems to the most recently developed DIV-BBBs, along with the advantages and disadvantages of each apparatus and technique. We have shown that static culture and coculture models (even though they represent cost-effective tools for drug permeability studies

17.8 Conclusion

Fig. 17.7 Future generation of DIV-BBB. The new apparatus features built-in electrodes for TEER measurements and multiplexing. The entire mechanism is computer-controlled.

across the BBB) cannot reproduce a fully functional BBB. We have also discussed the importance of blood flow as a critical modulator of EC differentiation and the functions that allow ECs to develop a morphology that closely mimics the endothelial phenotype in situ. This finding has brought researchers to develop a new generation of in vitro BBB models where quasi-physiological conditions for coculturing ECs and astrocytes are provided by capillary-like structure supports and the presence of intraluminal flow. These novel tools are able to functionally and anatomically mimic the brain microvasculature but have the disadvantage of being more expensive and requiring much higher technical skills. Expanding technologies will provide both researchers and pharmaceutical

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companies with cost-effective flow-based systems implemented with monitoring and analysis features, suited for both large-scale and laboratory use. The cell types currently used have also been discussed, setting an inevitable choice between fast data acquisition versus accuracy and reproducibility. We also want to direct attention toward the need for human cell-based models, since several pathological conditions cannot be reproduced in cell line-derived or rodentbased BBB models. Furthermore, with the rather novel prospect of stem cell research and expanding our understanding of the mechanisms behind cell-cell interactions and cross-communication at the BBB level, the future moves into a direction where differentiating factors will be successfully used to induce BBB properties in omnipotent stem cells.

Acknowledgments

This work was supported by NIH-2RO1 HL51614, NIH-RO1 NS 43284, NIHRO1 NS38195 to Damir Janigro and by The Alternatives Research & Development Foundation grant award to Luca Cucullo. We would also like to thank those who have in the past contributed to the development of the models described here: Liljiana Krizanac-Bengez, PhD, Kathe Stannes, BS, Eleonora Fornaciari, PhD, Patrizia Mascagni, PhD, Francesco Macchia, PhD MBA, and Francesca Salvetti, PhD.

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18 In Silico Prediction Models for Blood-Brain Barrier Permeation Gerhard F. Ecker and Christian R. Noe 18.1 Introduction: The In Silico World

Currently the drug discovery process starts with the identification of a drugable target followed by the establishment of a screening assay. The inhouse compound library is subsequently screened and a number of hits are identified. Standard high-throughput screening (HTS) libraries are composed of 100 000 up to 1 000 000 structurally diverse, drug/lead/hit-like compounds. Considering the fact that costs are from 0.09 to 0.9 $ per compound per screen and pharmaceutical companies screen up to 70 targets per year, HTS becomes rather expensive. Additionally, hit rates are rather low and are normally below 0.1%. Thus, in silico methods gain increasing interest in order to economize this process. In this context, several fields have to be considered: First of all, the proper design of combinatorial and HTS libraries requires tools for addressing chemical diversity and drug/lead/hit-likeness. Both issues are ongoing discussed in the literature and evolve continuously. For the calculation of diversity, numerous methods (based on distance, cells, fingerprints) and descriptors have been published. The assessment of druglikeness is strongly connected to the pioneering work of Lipinski [1]. However, the rule of five is often misinterpreted in terms of the prediction of bioavailability rather than a decision support system identifying compounds which might have problems with bioavailability. Since the work of Lipinski, numerous computational methods have been published addressing the drug-like/nondrug-like problem. Amongst them are the application of artificial neural networks [2, 3], recursive partitioning [4] and very recently also support vector machines [5]. Second, pre-screening filters for combinatorial libraries sorting out potential inactive compounds are increasingly used to reduce the number of compounds which have to be synthesized and screened by the simultaneous demand to keep all potentially actives. However, in recent years the trend has progressively moved away from a traditional single-target focus (by applying classic QSAR models) towards a family-based approach. Both structure-based target class-focused library design (e.g. for the protein kinase family) and ligand-based target Blood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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18 In Silico Prediction Models for Blood-Brain Barrier Permeation

class-focused library design (e.g. for the GPCR family) have been successfully applied [6]. Also the concept of privileged structures has been exploited within and across different target families and promises to be an effective approach to the discovery and optimization of novel bioactive molecules [7]. In this context, the computational methods applied have shifted from model generation tools to annotation, classification and data mining. Third, decision support systems addressing more complex issues such as phase I and phase II metabolism, blood-brain barrier permeation and toxicity are more or less routinely applied to further shape both the screening collection and combinatorial libraries. However, these issues are multifactorial, being mainly influenced by interactions with so-called nontarget proteins. Some of the key nontarget proteins responsible for poor ADME properties and/or high toxicity are the multidrug efflux pump P-glycoprotein (P-gp), the cytochrome P450 enzyme complex (CYPs) and the human ether-a-go-go related gene (hERG) potassium channel. All these proteins share a sort of promiscuity (or polyspecificity) in their binding interaction with ligands, which makes the use of rational drug design approaches to avoid undesirable drug-protein interactions rather difficult [8]. Especially in the case of blood-brain barrier permeation, several active transport systems are involved, which renders it rather difficult to properly predict brain uptake of drug candidates. This review intends to give an update on in silico models for the prediction of blood-brain barrier permeation, focusing on developments achieved within the past few years. For an overview on the literature before 2001, the reader is also referred to our recent review [9].

18.2 The Blood-Brain Barrier

The blood-brain barrier (BBB) separates the brain and central nervous system (CNS) from the bloodstream. Therefore, in CNS drug development it is of vital interest that the compounds are able to cross the BBB. Conversely, compounds designed for nonCNS targets should not cross the BBB, to avoid unwanted sideeffects. On principle, drug uptake into the brain may occur via five different routes or a combination thereof. Passive diffusion undoubtedly is the primary process for entering the brain. With respect to in silico methods, passive diffusion may be well described by physicochemical parameters of the respective solutes, such as lipohilicity, H-bond acceptor/donor features, molecular weight, polar surface area, number of rotable bonds. These descriptors can be calculated very easy and are thus a versatile tool for ultra high-throughput (HTS) in silico screening of large compound databases. Small hydrophilic molecules typically utilize the paracellular pathway to diffuse into organs. However, the main characteristic which distinguishes the BBB from intestinal blood vessels is the presence of high-resistance tight junctions between the brain capillary endothelial cells that form the barrier. This almost completely prevents the uptake of potential CNS drugs via the paracellular route and compounds passing the BBB al-

18.3 Data Sets Available

most exclusively have to utilize the transcellular pathway. However, in this case there is increasing evidence that, apart from passive diffusion, a broad variety of catalyzed transport systems are also involved. These include both carriermediated transport and receptor-mediated transcytosis for transporting compounds into the brain (active influx) and multidrug transport pumps for actively protecting the brain from unwanted chemicals (active efflux). In the case of transport into the brain, numerous systems have been discovered, including transport proteins for amino acids, monocarboxylic acids, organic cations, hexoses, nucleotides and peptides. Several of these proteins have been successfully used in prodrug strategies to enable or at least enhance brain uptake of neurotherapeutic agents. Classic examples are e.g. l-dopa and progabide. With respect to active efflux, the important role of ATP-binding cassette (ABC) pumps such as P-glycoprotein (P-gp) is increasingly recognized and first concepts to predict P-gp substrate properties of small compound series have been published recently. Undoubtedly, the BBB is designed to protect the brain from entering of toxic compounds. As outlined above, the main underlying concept seems to be to force the compounds to take the transcellular route, where nutrients are actively transported into the brain and possibly toxic compounds are expelled via active efflux pumps. Thus, BBB permeation is a multifactorial, complex issue which requires advanced computational methods for proper modeling.


In pharmacoinformatics, all attempts to develop predictive computational models heavily rely on the availability of suitable data sets. In QSAR analyses there are several recommendations, such as to span at least 2–3 orders of magnitude with the dependent variable, to guarantee identical modes of action for all compounds of the data set and to have at least 4–5 compounds per descriptor. Although methods used in classic QSAR studies developed rapidly and the use of algorithms for reduction of dimensionality, multivariate techniques and machine learning approaches allow a more generous handling of these rules, the requirement of proper data sets remains important. In the case of predictive models for BBB permeation, this raises some problems. First, as outlined in the previous chapter, permeation of the BBB is a multifactorial process and there is no distinct, single receptor to be addressed. Second, entrance into the brain is a dynamic process which, third, makes it difficult to mimic it in an appropriate in vitro assay system. Thus, the available data sets are very limited and clearly cannot be representative for the entire drug-like chemical space. On principle, three different types of data are available for in silico models. The most commonly used type of data are logBB values, which are defined as the ratio of the steady-state concentrations of a compound in the brain to those in the blood (Eq. 18.1). logBB = log([brain]/[blood])

18:1

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18 In Silico Prediction Models for Blood-Brain Barrier Permeation Table 18.1 Compounds used for in silico models of BBB permeation as given in [10]. Compound

LogBB

Compound

LogBB

Methane Pentane Hexane 2-Methylpentane 3-Methylpentane 2,2-Dimethylbutane Heptane 3-Methylhexane Cyclopropane Cyclohexane Methylcyclopentane Dichloromethane Trichloromethane 1,1,1-Trichloroethane Trichloroethylene 1,1,1-Trifluoro-2-chloroethane Halothane Teflurane Diethyl ether Divinyl ether Methoxyflurane Isoflurane Enflurane Fluorohexene Propanone Butanone Ethanol Propan-1-ol Propan-2-ol 2-Methylpropan-1-ol Benzene Toluene Ethylbenzene p-Xylene m-Xylene o-Xylene Cimetidin (1) a) 2 a) 3 a) 4 a) Salicylic acid Acetaminophen Ibuprofen

0.04 0.76 0.8 0.97 1.01 1.04 0.81 0.9 0.0 0.92 0.93 –0.11 0.29 0.4 0.34 0.08 0.35 0.27 0.0 0.11 0.25 0.42 0.24 0.13 –0.15 –0.08 –0.16 –0.16 –0.15 –0.17 0.37 0.37 0.2 0.31 0.29 0.37 –1.42 –0.04 –2.0 –1.3 –1.1 –0.31 –0.18

5 a) Clonidine (6) a) Mepyramine (7) a) Imipramine (8) a) Rantidine (9) a) Tiotidine (10) a) 11 a) 12 a) 13 a) 14 a) 15 a) 16 a) 17 a) 18 a) 19 a) 20 a) 21 a) 22 a) 23 a) 24 a) 25 a) 26 a) 27 a) 28 a) 29 a) 30 a) 31 a) 36 a) Y-G14 Y-G15 Y-G16 Y-G19 Y-G20 SKF 89124 SKF 101468 Acetylsalicylic acid Valproic acid Theophylline Caffeine Antipyrine Diazepam Phenytoin Hexobarbital

–1.06 0.11 0.49 1.06 –1.23 –0.82 –1.17 –2.15 –0.67 –0.66 –0.12 –0.18 –1.15 –1.57 –1.54 –1.12 –0.73 –0.27 –0.28 –0.46 –0.24 –0.02 0.69 0.44 0.14 0.22 0 0.89 –0.3 –0.06 –0.42 –1.3 –1.4 –0.43 0.25 –0.5 –0.22 –0.29 –0.05 –0.1 0.52 –0.04 0.1

18.3 Data Sets Available Table 18.1 (continued) Compound

LogBB

Compound

LogBB

Codeine Pentobarbital Alprazolam Indomethacin Oxazepam Hydroxyzine Desipramine Midazolam Promazine Chlorpromazine Trifluoperazin Thioridazine 32 a) 33 a) 34 a) 35 a) Mianserin Org4428 Org5222 Org16962 Org13011 Org32104 Org30526 Mirtazapine Tibolone Org34167 Risperidone Risperidone-9-OH Theobromine Morphine Propanolol Atenolol

0.55 0.12 0.04 –1.26 0.61 0.39 1.2 0.36 1.23 1.06 1.44 0.24 –0.34 –0.3 –1.34 –1.82 0.99 0.82 1.03 1.64 0.16 0.52 0.39 0.53 0.4 0.0 –0.02 –0.67 –0.28 –0.16 0.64 –1.42

Aminobarbital Phenylbutazone Aminopyrine Desmethyldesipramine Bretazenil Flumazenil RO19-4603 Paraxanthine Quinidine Salicyluric acid Fluphenazine Haloperidol Mesoridazine Sulforidazine Bromperidol Morthioridazine Nor-1-chlorpromazine Nor-2-chlorpromazine Desmonomethylpromazine Desmethyldiazepam 1-Hydroxymidazolam 4-Hydroxymidazolam Triazolam Clobazam Flunitrazepam Desmethylcobazam Thiopental Methohexital Didanosine Indinavir Nevirapine Zidovudine

0.04 –0.52 0 1.06 –0.09 –0.29 –0.25 0.06 –0.46 –0.44 1.51 1.34 –0.36 0.18 1.38 0.75 1.37 0.97 0.59 0.5 –0.07 –0.3 0.74 0.35 0.06 0.36 –0.14 –0.06 –1.3 –0.74 0 –0.72

a)

Chemical structures can be obtained from the authors on request.

Within the past decade, data from different sources have been collected and widely used for computational studies. However, when comparing the different sets used, it becomes evident that in total about 150 different compounds are present including also nondrug-like chemicals (Table 18.1) [10]. The most commonly used in vitro model is the Transwell system based on bovine brain microvascular endothelial cells (BBMEC). The system consists of a porous membrane support submerged in culture medium and is, therefore, characterized by a horizontal or vertical side-by-side diffusion system (Fig. 18.1). Co-culturing of these endothelial cells with primary astrocytes or the use of astrocyte-conditioned medium induces characteristics which are normally asso-

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Fig. 18.1 Schematic representation of the Transwell system.

ciated with the BBB, such as the presence of tight junctions and polarized expression of transport proteins. This leads to good correlations between in vitro and in vivo brain permeation data. However, there are several drawbacks which might be the reason for the lack of in silico studies utilizing permeability values based on data obtained from the Transwell system. First, most of the studies published are focused on technological and molecular biological issues. This implies that very often only small sets of structurally highly diverse molecules are investigated. The main question very often is to determine the tightness of the cell layer and to correlate the permeability of a given reference set of compounds to biological marker proteins. It normally does not address the pharmaceutical dimension of the problem, i.e. is there a statistically significant difference between structurally related compounds synthesized during a lead optimization program. Second, data published suffer from high inter-experimental variation which renders the permeability values rather noisy. This problem has been recently addressed by our group in order to generate data suitable for computational studies. In order to decrease inter-experimental variability, we simultaneously tested small groups of up to five compounds, including one reference compound (diazepam). Permeability values obtained were set in relation to the internal standard. This remarkably improved reproducibility and allowed the use of standardized logPE values as a dependent variable in regression analysis. The model obtained for a combined set of benzodiazepines and nonsteroidal antiinflammatory drugs showed a clear bilinear dependency of logPE values from calculated logP values (Fig. 18.2) [11]. This might become a standard method in our laboratory to generate BBB permeation data suitable for QSAR studies on homologous series of compounds. Several authors proposed a third method for generating data sets, especially for the use of large databases of known drugs. The underlying assumption of this approach is that compounds active on the CNS (CNS+) penetrate the BBB, whereas CNS– do not. While the first statement evidently is true, the second one is less reliable. Lack of CNS activity does not necessarily mean that a compound is not able to penetrate the BBB. It might also be the case that a CNS– compound is simply inactive against the respective molecular targets in the brain. This classification is thus suitable for developing predictive systems for CNS activity (yes/no), but cannot serve as basis for in silico BBB models.

logPEstand


MlogP Fig. 18.2 Bilinear dependency of standardized logPE values from the calculated Moriguchi logP for a set of benzodiazepines and NSAIDs [11].

To summarize, the major problem with regard to available data sets is the low number of compounds with accurately measured logBB values. Considering the fact that, via ChemNavigator, a screening library of almost 20 000 000 commercially available, drug-like compounds is offered, models based on at maximum 150 molecules are clearly not able to serve the whole chemical space. Additionally, almost no attention has been drawn to carefully monitor the main permeation driving/limiting force, i.e. passive diffusion, active influx or active efflux. Thus, the data available mostly represent the combined, overall picture and are thus only of limited value for in silico models which try to consider both passive and active transport phenomena.

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18.4 Computational Models

As outlined above, computational models have to consider two different categories of properties of the compounds under consideration. First, there are properties related to passive diffusion-controlled permeability, which are mainly influenced by the inherent physicochemical attributes of the compounds, such as logP, solubility and surface area (to mention just a few). For this, molecular descriptor-based methods have been used to generate predictive models. Second, we have to consider ligand-receptor, or in case of multispecific efflux transporters, ligand-antitarget interactions. These are normally targeted using pharmacophore-based approaches or classification systems. Obviously, a predictive in silico model suitable for both the lead identification and the lead optimization processes should include both categories. Although a broad, generic approach, which allows implementation of a single software tool for reliable prediction of BBB permeation taking into account passive and active phenomena, would be the optimum, until now this seems beyond reality.

18.5 Passive Diffusion 18.5.1 Regression Models

Although available data are rather limited, a number of in silico BBB models have been published in the literature. Most of them are based on classic regression equations. In the early 1980s, Levin published a correlation between the brain capillary permeability coefficient and log(P[MW]–1/2) for molecules with MW < 400 (Eq. (18.2). logPc 0:4115 logPMW

1=2

4:605

18:2

r 0:91; n 22 Despite being one of the first QSAR studies on BBB permeation data, the most interesting items are the four outlyers adriamycine, epipodophyllotoxin, vincristine and bleomycine (Fig. 18.3). All three compounds showed lower permeability coefficients than expected from their log(P[MW]–1/2) values. The author tried to rationalize this behavior by proposing that the compounds due to their high lipophilicity may penetrate and distribute into – but not through – the brain capillary endothelia. Nowadays, we know that all these natural product toxins are excellent substrates for P-gp. Later on, more advanced regression models were published using additional descriptors such as DlogP, lipoaffinity, polar and accessible surface area, polariz-

18.5 Passive Diffusion

Fig. 18.3 Protein homology model of P-glycoprotein. Red and yellow: regions of highest labeling with propafenone-type photoaffinity ligands [53].

ability, free energy of solvation and H-bonding properties. Although the octanol/water partition coefficient plays a predominant role in many regression models, in some cases it shows only a weak correlation with logBB. Ter Laak et al. found that the brain permeability of a series of structurally diverse histamine H1 receptor antagonists was better explained by logD rather than by logP, which also takes account of the dissociation properties of the respective compounds. Kaliszan included the molecular weight as additional descriptor for molecular bulkiness (Eq. 18.3). logBB 0:272DlogP

0:00112Mm

0:088

n 33; r 2 0:90; s 0:126; F 131:1

18:3

411

412


Taking into account also descriptors related with molecular surface properties, both Kelder (Eq. 18.4) and Clark (Eq. 18.5) published equations using the polar surface area (PSA) as the only descriptor. logBB 1:33

0:032 PSA

18:4

n 45; r 2 0:84; F 229 logBB 0:55

0:016 PSA

18:5

n 55; r 2 0:71; s 0:41; F 128 However, the drawback of using PSA alone is the inability to distinguish the differences of hydrocarbon compounds. Thus, Clark combined both hydrophobicity and PSA, which improved the model (Eq. 18.6). logBB 0:152 ClogP

0:148 PSA 0:139

18:6

n 55; r 2 0:79; s 0:35; F 95:8 More complex models include the dipolarity/polarizability parameters, calculation of free energy values and the solvent-accessible surface area (SASA) calculated via Monte Carlo simulations of compounds in water. This approach is based on the assumption that the solvation of compounds in water and a lipid phase might be accompanied by conformational changes, which will be more pronounced for large flexible molecules. In an ideal setting, one might perform a simulation of the diffusion of molecules through a lipid bilayer, applying molecular dynamics simulations. Unfortunately, the timescales of diffusion of small molecules are in the range of several microseconds, whereas MD simulations normally only span a few nanoseconds. Therefore, in an extension of the work of Jorgensen, Kaznessis et al. used Monte Carlo simulations of a set of 85 compounds in water to calculate diffusion-related properties, such as the SASA, the hydrophobic, hydrophilic and aromatic component of SASA, the dipole moment and both the Coulomb and Lenard-Jones energies between solutes and solvent. Although several highly predictive models were obtained (r = 0.97), it has to be noted that computation times were rather high (1.6 CPU hours per compound on a 600 MHz Pentium PC). Hutter used information derived from quantum chemical calculations for a model based on a training set of 90 compounds [12]. In total, 41 descriptors were calculated using the semiempirical VAMP program package. In addition to several atom counts, the number of ionizable groups, number of aromatic rings and number of H-bond donors and acceptors, a set of descriptors which account for the molecular shape and expansion of the respective molecules were also used (PCGA, PCGB, PCGC). These descriptors are derived from a principal component analysis (PCA) of the molecular geometry and are computed as the square root of the principal moments of the cartesian coordinates. The first principal component (PC)


corresponds to the largest extension of the molecule, the second PC to the largest extension perpendicular to the first PC and the third PC to the largest extension perpendicular to the first and second PC, respectively. The magnitude of these dimensions is assumed to be related to the likelihood of penetrating the membrane. The final equation obtained had a r2 value of 0.865 and contained several countbased descriptors (H-bond donors, halogens, NO2, sulfur atoms, six-membered aromatic rings, rotable bonds) and shape descriptors PCGA and PCGC, as well as quantum chemically derived ones. These include values based on the molecular electrostatic potential and the covalent hydrogen bond basicity. Outlyers were shown to underlie the active transport processes. Computation time for the whole data set was about 3 h on a single CPU in a Compaq Alpha Server ES40 (667 MHz), which corresponds to an average of one molecule per minute. Iyer et al. followed an even more demanding computational approach in predicting BBB partitioning using membrane interaction QSAR analysis (MIQSAR) [13]. In MI-QSAR, structure-based design methodology is combined with classic ligand-based, descriptor-driven QSAR analysis to model chemically and structurally diverse compounds interacting with cellular membranes [14, 15]. In the present study, a training set of 56 compounds was used and molecular dynamics simulations were performed to determine the explicit interaction of each test compound with a model DMPC monolayer membrane model. This gave a series of solute-membrane interaction descriptors. Additionally, a series of dissolution and solvation solute descriptors (aqueous solvation free energy, 1octanol solvation free energy, logP, cohesive packing energy of solute molecules, hypothetical crystal-melt transition temperature, hypothetical glass transition temperature of the solute) and a set of 18 general intramolecular solute descriptors, such as HOMO, LUMO, dipole moment, molecular volume, molecular surface area, MW and PSA, were also calculated and combined with the membrane-solute descriptors using multiple linear regression (MLR) analysis and genetic function approximation (GFA). Validation of the models was performed both using Y-scrambling and an external test set. Judged on predictive power q2, the best model obtained included five descriptors (Eq. 18.7). logBB 0:1591 ClogP 0:0231 PSA 0:0071EMS chg-hbd 0:0346ESS tor 0:0075DETT 1 4 0:0156

18:7

n 56; r 2 0:845; q2 0:795 EMS(chg-hbd) represents the total intermolecular electrostatic and hydrogen bonding interaction energy between the solute and the DMPC monolayer. ESS (tor) is the torsion energy of the solute for the solute located at the position corresponding to the lowest solute-membrane interaction energy state of the model system. DETT(1–4) is the change in the 1,4-nonbonded interaction energy of the system due to the uptake of the solute from free space to the position corresponding to the lowest solute-membrane interaction energy state of the model

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system. According to the logBB MI-QSAR models obtained, the authors proposed the following physicochemical factors influencing BBB permeation. · The relative polarity of the solute, as represented by PSA and logP. In general, less polar, more lipophilic compounds partition more readily into the membrane. · The strength of interaction (binding) of the solute with the membrane. The greater the binding, the higher the BBB partitioning. · The conformational flexibility of the solute in the membrane and the conformational flexibility of the membrane-solute complex. Increasing flexibility corresponds to increasing logBB values. Unfortunately no information on CPU time needed is given, so this very interesting approach cannot be judged on the basis of computational costs. Lobell et al. followed two different lines, one suitable for a highly accurate prediction in a low throughput mode and another optimized for ultra-highthroughput processing [16]. For the first one, they calculated 34 descriptors, such as logP, molar refractivity (MR), desolvation energies in water and octanol, the total polar surface area, MW, number of rotable bonds, H-bond donors and acceptors, kappa indices, several indicator variables for charge and ten so-called shadow descriptors. These are a set of geometric descriptors that help to characterize the shape of molecules. Shadow descriptors are calculated by projecting the molecular surface on three mutually perpendicular planes, thus signifying the ratio of the largest to the smallest dimension. The coefficient of these descriptors in the final equation indicated that spherical shapes have a small advantage compared with rod-like shapes, with regard to transport across the BBB. To capture the charge influences, predicted pKa values were used to classify the compounds depending on the pH range when these groups obtain a predominantly positive or negative charge. This is based on Seelig et al., who have already shown that compounds that contain an acidic group with pKa < 4 or a basic group with pKa > 10 do not cross the BBB by passive diffusion [17]. Thus, Lobell et al. defined six classes and compounds were either assigned 0 or 1 for each class. However, this was done manually which renders the method rather slow. For high-throughput in silico calculations, the C2-ADME logBB module implemented in the Cerius2 software package was used. Comparing the results with other established methods, such as those published by Lombardo, Feher, Clark, Kelder and the authors themselves, proved the accuracy of the models. Hou and Xu applied a genetic algorithm (GA) to a large set of descriptors and published a model for a set of 96 structurally diverse compounds [18]. Taking into account the difficulty in selecting the appropriate descriptors for a problem as complex as permeation through the BBB, GAs might be the method of choice. GAs are expected to find a group of reliable QSAR or QSPR models from a large number of samples very efficiently. This method was first proposed by Rogers and Hopfinger [19] and Kubinyi [20]. Using 27 descriptors, 100 models with three descriptors and 100 models with four descriptors were obtained. The best model is shown in Eq. (18.8).


logBB 0:32 Dlog P 0:11 RotBonds 0:0024 Jurs-PNSA-2 0:35RadofGyration 0:62

18:8

n 59; r 0:87; q2 0:84; sd 0:41; F 42:14 In an extension of this work and taking into account the importance of logP for BBB models, the authors introduced SLOGP, a new parameter for lipophilicity. SLOGP is based on simple atomic addition and thus quickly and efficiently calculates lipohilicity values. The values obtained by SLOGP correspond well to those from other algorithms, such as CLOGP, ALOGP, ALOGP98 and HINT. The generation of several models via stepwise inclusion of additional descriptors, such as the highly charged polar surface area (HCPSA) and a parameter for molecular bulkiness (MW-360) yielded the highly significant Eq. (18.9): logBB 0:197 SLOGP 0:0135 HCPSA 0:0140 MW 360 0:00845

18:9

n 78; r 0:876; s 0:364; F 81:5 HCPSA reflects the polar surface area given by polar atoms with absolute partial charges larger than 0.1 as calculated according to Gasteiger. Use of (MW-a) introduces a special spline model for bulkiness. Thus, if the molecular weight (MW) is lower than a given value a, the term (MW-a) is set to 0. Otherwise the term is used as calculated. This regression with splines allows the incorporation of features that do not have a linear effect over their entire range. To determine the best value of a, a systematic search was used changing this value from 100 to 400 using a step of 10. The best equation was obtained for a = 360. The use of these three descriptors gives a meaningful physical picture of the molecular mechanisms involved in BBB permeation. First, hydrophobic molecules can permeate the blood-brain barrier more easily than hydrophilic ones. Second, the larger the polar surface area is, the more negatively this accounts for BBB permeation. However, this contribution is limited to those atoms with high charge densities (HCPSA). Third, large molecules also will show limited BBB permeation, but this bulk effect may become effective only when the MW is higher than 360. To improve efficiency and speed of calculation, the authors also reparameterized the SLOGP algorithm and developed a new set of parameters to calculate a topological highly charged polar surface area (HCTPSA). This allows the prediction of logBB values solely on basis of the topological structure of the molecules and thus renders the method a high-throughput logBB prediction method. Corresponding software packages are available on request from the authors. A completely different, but also atom-based approach was published by Sun. In a search for a generic molecular descriptor system which allows the prediction of most of the relevant properties of a molecule (logP, solubility, logBB, intestinal absorption), the author developed an atom classification system. First, a primary classification tree based on experience and chemical intuition was im-

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plemented using the Daylight Toolkits. Then, the logP data set from Starlist was used to determine more accurately where to split and where to stop splitting the tree. The Starlist contains 10 974 compounds which span a logP range of 16 orders of magnitude and include chemical entities from simple alcohols to complex peptides. The atom types identified from the original classification tree served as molecular descriptors to generate the predictive model for logP calculation. Thorough analysis of the errors and standard deviations of the respective coefficients yielded clues for further modification of the classification tree. Repeated optimization cycles finally led to a final model for the calculation of logP values with a r2 of 0.912. In total, 218 atom types were identified, comprising 88 types of C, seven types of H, 55 types of N, 31 types of O, eight types of halides, 24 types of S, six types of P and 26 correction factors (no aromatic rings, intramolecular hydrogen bond, etc.). For the BBB permeation model, the data set of Abraham (57 compounds) was used and a total of 94 different atom types were identified for this data set. Additionally, half of these atom types occurred only once or twice in the data set. This imbalance in the number of descriptors to number of cases made it difficult to properly calculate the contribution factors for each atom type. Thus, the predictive power obtained in leaveone-out cross-validation runs did not exceed 0.55. Also r2 values for an external prediction set were rather low, but could be improved when increasing the structural diversity of the training set by the incorporation of additional compounds. The author thus concluded that “a better predictive model should be derived, once a larger data set of high quality becomes available”. A complete different set of descriptors was used recently by Rose and Hall, who published a model based on the electrotopological state indices. In the E state representation, the central feature is the intrinsic state term Ii, which encodes in an integrated fashion both electronic and topological attributes. Thus, E states somehow represent the electronic distribution across a molecule which forms the basis for both steric and electronic properties of the molecule. For a set of 102 structurally diverse compounds, use of the hydrogen E state index for hydrogen bond donors [HST(HBd)], the hydrogen E state index for aromatic CHs [HST(arom)] and the second-order difference valence molecular connectivity index (d2vv) gave a model with reasonable predictive power [q2 = 0.62; Eq. (18.10)]. logBB 0:202 HST HBd 0:00627 HST arom2 r 2 0:66; s 0:45; F 62:4; n 102; q2 0:62

0:105 d2 vv 2

0:425 18:10

Detailed analysis of the model indicates that molecules with large HST(arom) values (presence of aromatic rings), small HST (HBd) values (few and weak Hbond donors) and low d2vv values (less branched molecules with few electronegative atoms) are able to penetrate the BBB. Despite cross-validation and the use of an external test set selected randomly from the data set available, an ex-


ternal prediction of the CNS± data set of Crivori et al. was also performed. The model correctly classified 27 out of 28 molecules. Furthermore, logBB values in a large database of drug-like molecules (20 039) were computed to demonstrate the general applicability of the approach to the drug-like chemical space. In their work, Pan et al. argued that a common mechanism of action, which is one of the most basic assumptions of QSAR analysis, may not be satisfied for ADMET data sets [21]. Thus, structurally diverse compounds might interact in different modes with the membrane. To overcome this problem, the authors divided the whole data set of 150 compounds into subsets based on 4D molecular similarity (4D-MS) measures. Subsequently, predictive QSAR models were constructed for each cluster subset using the already described MI-QSAR descriptors derived from membrane interaction simulations. 4D-MS developed by Hopfinger and co-workers also takes the thermodynamic distribution of different conformers of a given molecule into account [22]. Thus, a conformation ensemble profile of each member of a set of molecules is generated using molecular dynamics simulations. This is followed by construction of the main distancedependent matrix (MDDM) for each pair of interaction pharmacophore elements (IPE) of each molecule. Subsequent PCA transforms the MDDM into a set of eigenvalues which are normalized and sorted. This set of normalized eigenvalues is used as a fingerprint for each molecule and serves as basis for the clustering of the compounds into structurally related subsets. In the current study, the set of 150 compounds was divided into three subsets. The optimized QSAR model for the complete data set is similar to the model reported by Clark and includes ClogP and TPSA as descriptors (Eq. 18.11). logBB 0:20 ClogP

0:01T PSA 0:064

18:11

n 150; r 2 0:69; q2 0:60 Subset 1 includes 37 compounds which are mainly simple, nondrug-like molecules with less than two heteroatoms (excluding halogens). Dividing the subset into a training and test set and performing a multiple linear regression analysis gave Eq. (18.12), in which Echg represents the electrostatic interaction energy for the solute-membrane complex. logBB 0:26 ClogP 0:00077 Echg 0:26

18:12

n 24; r 2 0:85; q2 0:83 For subset 2, which contains 88 compounds (63 training, 25 test), in addition to PSA and ClogP, the two topological descriptors S_sF and Kappa3 also showed statistically significant contributions Eq. (18.13). S_sF is the electrotopological state index for a fluorine atom with one single bond and Kappa3 is generally considered an index which reflects the molecular shape of a molecule.

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logBB 0:25 ClogP

0:01 PSA

0:025S sF

0:11 Kappa3 0:66

18:13

n 63; r 2 0:69; q2 0:66 Interestingly, in subset 3, which contains 25 compounds (17 training, 8 test), ClogP is not significant in explaining logBB values (Eq. 18.14). Instead, the two descriptors Ecoh and DETT(vdw) are found in its place. Ecoh is the cohesive energy of the solute, which reflects how well a compound self-aggregates. DETT(vdw) is an explicit MI-QSAR descriptor of the van der Waals energy difference between the solute-membrane complex and the free states of the solute molecule and the membrane monolayer. logBB 0:069Ecoh

0:095 DETT vdw

0:031 PSA

0:58

18:14

n 17; r 2 0:80; q2 0:72 To better evaluate the advantage of dividing the whole data set into three subsets, a QSAR model of the combined training sets was also performed. The optimum model for this overall training set is shown in Eq. (18.15): logBB 0:19 ClogP

0:011 TPSA 0:05

18:15

n 104; r 2 0:69; q2 0:64 As exemplified by the statistical parameters, each of the three models for the subsets showed a higher predictive power (q2) than the overall model. A more detailed analysis showed that the general model captures almost the same information as submodels 1 and 2, whereas new, additional information is provided by model 3. Overall, it appears to be advantageous to build individual QSARs for subsets of large data sets, because “minority” descriptors which are only descriptive for a small subset of compounds do not survive in a general model built for the whole data set. This might be an excellent approach to deal with the different mechanisms influencing BBB permeation, such as passive diffusion and active influx/efflux. Last but not least the recently introduced VSA descriptors have to be mentioned. These descriptors are based on the atomic contributions of logP (10), molar refractivity (8) and partial charge (14) to the van der Waals surface area. In total this yields 32 descriptors which were used to build models for boiling point, vapor pressure, free energy of solvation in water, solubility in water, thrombin/trypsin/factor Xa activity and BBB permeability [23]. For the latter, the data set of Luco was used. Principal component regression analysis led to a final model based on 15 descriptors, which showed a remarkably high r2 value (0.83).


18.5.2 Classification Systems

In the early stages of drug discovery, there is no need for highly sophisticated prediction tools for logBB values. These are definitely advantageous in the lead optimization process, where the compound of interest has already a given pharmacological activity and BBB permeation. In the discovery of new lead compounds, high-throughput in silico tools are favorable which give early alerts to the medicinal chemist, thus guiding both the design of combinatorial libraries and the acquisition of compounds for the HTS library. For this purpose, simple descriptors such as atom counts or easy-to-calculate physicochemical parameters seem to be preferred over methods which require the calculation of interaction fields or quantum chemical parameters. Undoubtedly the pioneering work of Lipinski paved the way for rule-based methods for assessing drug-likeness, leadlikeness or even hit-likeness. Thus, in analogy to the classic rule of five, several rule-based methods for BBB permeation have been published. Norinder and Haeberlein proposed a very simple system based on only two rules: 1. If the number of N + O atoms in a molecule is five or less, then it has a high chance of entering the brain. 2. If ClogP–(N + O) > 0, then logBB is positive. These rules are based on the fact that there is a strong correlation between the number of nitrogens and oxygens and the respective PSA value. The polar surface area has already been recognized as a versatile descriptor by both van de Waterbeemd and Kelder, using an upper limit for PSA to assess the likelihood of brain uptake. Analyzing 125 marketed drugs, van de Waterbeemd proposed upper limits for PSA (90) and molecular weight (450). Extending the training set to compounds being at least in clinical phase II studies, but not on the market yet, Kelder et al. found a slightly lower limit for PSA (60–70). For classification purposes, data sets used are very often clustered into CNS active and nonactive drugs. This leads to a remarkable increase of the number of compounds available for training and testing. However, one has to bear in mind that it is not necessarily true that nonCNS drugs are not entering the brain. While CNS+ automatically includes BBB+, CNS– might also be BBB+. Nevertheless, several authors followed this approach and several interesting models have been published, mainly using artificial neural networks (ANNs) for classification. ANNs are robust to noise and, once trained, allow fast and accurate predictions. Thus, both Sadowski and Kubinyi [24] and Ajay et al. [25] presented networks which are able to distinguish between drugs and nondrugs. Molecules are encoded using Ghose-Crippen descriptors and the networks are used for in silico screening of combinatorial libraries prior to synthesis and for analysis of commercially available libraries. An identical approach was applied by Ajay et al. to distinguish between CNS and nonCNS drugs [26]. The authors used a Bayesian neural network, which is known to be robust to noise introduced by misclassifications. This is of utmost importance, because the assign-

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ment whether a given compound is regarded as CNS+ or CNS– was made solely on the basis of the therapeutic use of the compound. As outlined above, this approach may lead to a lot of misclassifications in the CNS– group. Additionally, CNS– does not necessarily mean BBB–, so this network should not be used for predicting BBB permeation behavior. However, the network correctly predicted 80% of a test set comprising 13 000 CNS+ and 53 000 CNS– compounds from the databases used for verification. Recently, Engkvist et al. presented an in silico ADMET prediction tool based on substructural analysis [27]. Substructural analysis has a long history and is based on the rather simple principle that the pharmacokinetic properties of a molecule depend on its chemical structure, i.e. atom types and topological information. Thus, it should be possible to assign a molecule with a specific fragment composition to a certain cluster (i.e. CNS+) by analyzing the distribution of molecular fragments within a large data set with known CNS activities. For training, the authors divided the World Drug Index (WDI) into three groups: (1) drugs that must pass the BBB for their action (psychosedatives, psychostimulants, antidepressants), (2) drugs that might pass the BBB (antiemetics, antiparkinsonians) and (3) drugs that most likely do not pass the BBB (cytostatics, antibiotics, antihypertensives). Class (1) contained 3678 molecules and was designated as CNS+, class (2) was omitted and, out of the 50 025 molecules from class (3), 5000 were randomly selected to serve as CNS– compounds. Models obtained with the SUBSTRUCT software package were able to separate CNS active and nonactive compounds with approximately 80% accuracy. Analysis of the model showed that protonated nitrogens, aromatic rings, chlorine and fluorine are more frequent among CNS active compounds, while the opposite is true for oxygen-containing compounds. In comparison to an ANN, the ANN was faster (2 s for 1000 molecules) than SUBSTRUCT (18 s) in classifying unknown compounds, but did not allow to identify the crucial features for separating CNS+ and CNS– compounds. Adenot and Lahana used discriminant analysis (DA) and PLS-DA to develop a model which is able to filter CNS drugs from large virtual libraries, taking into account simultaneously both passive diffusion and efflux transport components [28]. After carefully analyzing the WDI, they ended up with a heterogenous CNS library of approximately 1700 compounds, including 1336 BBB+ drugs, 259 BBB– drugs and 91 P-gp substrates. Out of a large set of descriptors combining molecular properties, surface areas, electronic parameters and topology, DA identified a small set of simple descriptors as being sufficient to evaluate BBB permeation in most cases. Thus, the number of heteroatoms is sufficient to predict BBB permeation with a high rate of well classified compounds (92%). All compounds were characterized by both a passive diffusion component and a P-gp efflux component. Combining these two components allowed mapping of the CNS compounds. This mapping clearly delineates three distinct areas, i.e. P-gp+ drugs, BBB+ drugs and BBB– drugs. Interestingly, several CNS drugs, like amitryptiline, chlorpromazine, morphine and disulfiram, are also P-gp substrates in vitro, but have been predicted with low P-gp activity. This is somewhat

18.6 Field-Based Methods

contradictory and indicates the complexity of the in vivo system. Additionally it has to be stressed that, in the case of P-gp substrates, properties are difficult to measure. Using inside-out membrane vesicles from P-gp-expressing cells Schmid et al. could demonstrate that some propafenone-type P-gp inhibitors stimulate the ATPase activity of P-gp [29]. This indicates that inhibitors might be substrates which block the pump via a rapid rediffusion process. In this case, no net transport is observed and the pump is kept busy, which gives the macroscopic picture of being blocked. With the armory of supervised machine learning approaches, support vector machines (SVMs) have been introduced as a powerful, theoretically well founded algorithm, capable of dealing with large, high-dimensional, nonlinear classification problems [30]. A particularly important feature of SVMs is that they explicitly rely on statistical learning theory [31] and thus avoid overfitting. The key concept is the structural risk minimization principle proposed by Vapnik and Chervonenkis [32]. SVMs have consistently achieved a performance competitive with ANNs, especially in the field of ADME property classification. They have been used for compound classification in several stages of the drug discovery process, including the drug/nondrug problem [33] and prediction of BBB permeation. For the latter, the data set implemented in VolSurf was used, which is the result of in vivo studies and contains 337 BBB+ and 139 BBB– compounds [34]. Applying the 72 VolSurf descriptors, a SVM based on a radial basis function outperformed ANNs as well as a C5.0 decision tree and a nearest-neighbor classifier, showing an overall accuracy of 0.87 [35].

18.6 Field-Based Methods

Field-based methods are gaining an increasing interest in the in silico modeling of complex phenomena. In the VolSurf approach recently introduced by the group of Cruciani, molecular interaction fields computed by GRID [36] are used for calculation of a set of descriptors. Thus, compounds are placed in a 3D grid with a given distance between grid points and so-called probes are placed on the grid points. Then the steric, electrostatic, hydrophobic, H-bond acceptor and H-bond donor interaction energy between the probe and the compound is calculated. The probes are designed to mimic protein-ligand interaction forces and include, e.g., H+, carbonyl, DRY (methane), aromatic ring and tyrosine-OH. This interaction energy matrix is subsequently used to extract a set of 72 descriptors. These so-called VolSurf descriptors refer to molecular size and shape, the size and shape of hydrophobic and hydrophilic regions, hydrogen bonding, amphiphilic moments and critical packing [37]. These descriptors have been shown to be very useful for the modeling and prediction of pharmacokinetic properties [38]. For the BBB model, the authors used the water, DRY and carbonyl oxygen probes for calculating the interaction energies of 110 compounds. Principal component analysis led to a model which was able to separate BBB+

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from BBB– compounds quite well and showed correct classifications of 90% of the BBB+ and 65% of the BBB– compounds in an external test set of 120 compounds. In a further refinement of the model, the combined data set (229 compounds) was used and a categorial score of +1 was given to BBB+ compounds and –1 to BBB– and BBB± compounds. Subsequent PLS analysis gave a model which distinguishes well between BBB+ and BBB– compounds [37]. The coefficient plot revealed that descriptors for hydrophilic regions, capacity factors, Hbonding and polar water accessible surface area are inversely correlated with BBB permeation, whereas those encoding hydrophobic interactions are directly correlated with BBB permeation. In the course of our studies, we projected both a set of benzodiazepines and a series of polyamines on the pre-defined BBB model. The benzodiazepines are all classified as BBB+, which is quite obviously in accordance to their pharmacological profile as CNS drugs. However, the plot of the polyamines shows that they cover a new chemical space (Fig. 18.4). The same was in part true for a set of glycine-antagonists we are currently testing on our in vitro BBB model. Thus, the results have to be taken with caution and an extension of the training set with focus on a higher structural diversity is highly recommended. As mentioned already several times, 100–200 molecules are by far too few to allow in silico models with general predictivity. In a recent paper, the VolSurf approach was extended to yield a quantitative model based on logBB values of 83 compounds [39]. A four-component PLS model was obtained which, after removal of four outlyers, showed a q2 value of 0.65. Analyzing the contribution of each descriptor the following conclusions were drawn: 1. Descriptors of polarity (hydrophilic regions, capacity factors) are inversely correlated with logBB values. 2. Descriptors for hydrophobic interactions are directly correlated to BBB permeation. 3. Descriptors for size and shape have no pronounced effect on brain uptake. Although the VolSurf approach is a very powerful tool not only for predicting BBB permeation behavior, but also for modeling ADME parameters, several drawbacks have to be considered. First, VolSurf is a 3D-QSAR method, which implicates that it is conformation-dependent. Although it has been shown that the influence of different conformations within, e.g., homologous series of molecules is relatively low, we observed deviations in q2 values in the range of 0.1 when using different methods for the generation of 3D structures from 2D-sdf files. Second, it is relatively time-consuming and calculation of the descriptors for a set of 10 000 molecules needs about 2 days on a Pentium 2 MHz Linux PC. Third, the descriptors derived are rather abstract and information on how an optimal molecule should look are difficult to retrieve. However, VolSurf represents an interesting approach to derive predictive models for complex types of biological phenomena and models for P-gp, cytochromes and the human ethera-go-go-related gene potassium channel (hERG) are on the way.

18.7 Active Transport

Fig. 18.4 Projection of a series of polyamines (yellow) onto the VolSurf model provided by Tripos (www.tripos.com). Red: BBB+, blue: BBB–.


In vivo, transcellular transport of compounds into the brain is mediated either via passive diffusion or by carrier transport and receptor transcytosis. With the increasing knowledge on the transport processes involved in brain uptake, a series of efflux transport proteins have also been identified, such as the multidrug transporter P-glycoprotein (P-gp) and the organic anion transport protein (OATP) family of transporters. These proteins function as gate-keepers, thus preventing the entry of toxic compounds into the brain [40]. Most of them show a broad substrate specificity coupled with a high efficiency. Undoubtedly, active transport is an important functional characteristic and in silico prediction methods have to take this into account. However, due to its original discovery as a main contributor to multiple drug resistance in tumors, research on P-gp focused on the development of inhibitors rather than the prediction of substrate

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properties [41–43]. Nowadays, the role of P-gp in ADME and especially brain uptake [44] is well understood and thus P-gp is increasingly considered as an antitarget [45–47]. Within the past few years, several models have been published for the prediction of P-gp substrate properties. Didziapetris et al. developed a classification system based on simple descriptors. Based on a set of 220 compounds with data from polarized transport across MDR1 transfected cell monolayers, the authors derived the “rule of fours” for a crude estimation of P-gp substrates [48]: 1. substrates: (N + O) ³ 8, MW > 400, acid pKa > 4 2. nonsubstrates: (N + O) £ 4, MW < 400, base pKa < 8 These rules are based on the compound size, H-bond accepting properties and ionization; and they support the view that P-gp functioning can be compared to a complex system with fuzzy specificity. Gombar and colleagues built a QSAR computational model that predicts the outcome of their inhouse monolayer P-gp efflux assay. Based on a training set of 95 compounds, a two-group linear discriminant model has been developed, which shows a sensitivity of 100% and a specificity of 90.6%. The 27 descriptors used mainly account for the ability to partition into membranes, molecular bulk and the counts and electrotopological values of certain hydrides. Within this work, several simple relationships have also been derived. The most striking one is the relationship between the molecular E state (MolES) and the P-gp substrate classifier (0 or 1). Among the 95 training set molecules, those with MolES > 110 were predominantly P-gp substrates (95.0%) and those with MolES < 49 were nonsubstrates (84.6%). This “Gombar-Polli rule” may be a rapid way to initially screen large combinatorial and virtual compound libraries [49]. The challenging task of predicting P-gp substrates also served as a model case to prove the accuracy of support vector machines. In total, 159 descriptors comprising 18 descriptors in the class of simple molecular properties, 28 descriptors for molecular connectivity and shape, 84 electrotopological state descriptors, 13 descriptors in the class of quantum chemical properties and 16 descriptors for geometrical properties were used as the input vector [50]. The SVM model obtained gave a prediction accuracy of 81% for P-gp substrates and 79% for nonsubstrates. This is slightly better than the performance obtained with k-nearest neighbor, ANNs and a C4.5 decision tree. Extending their CATALYST pharmacophore model for P-gp inhibitors, Ekins et al. generated a pharmacophore model for P-gp substrates. Using a set of 16 inhibitors of verapamil-binding to P-gp, the authors generated a pharmacophore that consisted of one hydrogen bond acceptor, one ring aromatic feature and two hydrophobes [51]. This model correctly predicted the rank order of the four data sets used for the inhibitor models and also fit the three substrate probes verapamil, vinblastine and digoxin. This indicates the presence of partially overlapping binding sites within P-gp. Very recently, Pleban et al. used a combined photoaffinity labeling-protein homology modeling approach to generate a 3D model of P-gp. Briefly, a series


Fig. 18.5 Proposed workflow for combined BBB permeation models. (Modified from Ref. [54]).

of propafenone-type benzophenones were synthesized and used as photoaffinity probes to map the binding regions of P-gp. After UV irradiation in the presence of the benzophenone ligands, the protein was isolated and digested. Subsequent MALDI-TOF mass spectrometry revealed a series of fragments which were covalently labeled by the photo probes [52]. Generation of a protein homology model on the basis of the recently published X-ray structure of the ABC transporter MsbA from Vibrio cholerae and projection of the regions with the highest labeling frequency showed two distinct regions involved in ligand binding. Labeling is predicted by the model to predominantly occur at the two transmembrane domain/transmembrane domain interfaces formed between the amino- and carboxy-terminal halves of P-gp. These interfaces are formed by transmembrane (TM) helices 3 and 11 on the one hand and TM segments 5 and 8 on the other hand (Fig. 18.3) [53]. After subsequent molecular dynamic simulations, these model may serve as a versatile tool for docking experiments and target-based in silico screening of compound libraries.

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18.8 Conclusions and Future Directions

Although great progress has been made in the development of in vivo and in vitro models for BBB permeation, their throughput is still rather limited. In silico methods can help to establish predictive rules or equations for high-throughput permeability prediction as a first screen in the drug discovery process. However, with the increasing knowledge of the numerous active transport processes involved, it is becoming evident that models based on passive diffusion alone are not suitable to cover the whole drug-like chemical space. First, hybrid (in vitro/ in silico) models capable of predicting both passive and transporter-mediated contributions appeared in the literature and several models for predicting P-gp substrate properties have been described. As outlined in a recent technological report, a combination of techniques is recommended to cover the whole range of drug entry and efflux mechanisms. Combining serial and parallel screening processes may guide the selection of the right hits and facilitate the ranking of lead compounds (Fig. 18.5) [54]. The outcome of an expert system which combines different models undoubtedly will be greater than the sum of its parts.

Acknowledgment

We gratefully acknowledge financial support by the Austrian Science Fund, grant P14582-N03.

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Part V Drug Delivery to the Brain


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19 The Blood-Brain Barrier: Roles of the Multidrug Resistance Transporter P-Glycoprotein Sandra Turcotte, Michel Demeule, Anthony Régina, Chantal Fournier, Julie Jodoin, Albert Moghrabi, and Richard Béliveau

19.1 Introduction

For the brain, the blood-brain barrier (BBB) formed by the brain capillary endothelial cells (BCEC) is considered to be the major route for the uptake of endogenous and exogenous ligands into the brain parenchyma [1]. The EC of brain capillaries are closely sealed by tight junctions and constitute a continuous endothelium. Moreover, brain capillaries possess few fenestrae or endocytic vesicles as compared to the capillaries of other organs [1–3]. BCEC are surrounded by astrocytes, pericytes, microglial cells and by the extracellular matrix. The close association of BCEC with the astrocyte foot processes and the basement membrane of capillaries is important for the development and maintenance of the BBB properties that permit tight control of the blood-brain exchange of molecules [1–4]. The restrictive nature of the BBB is due, in part, to the tight junctions that prevent significant passive movement of small hydrophilic molecules between blood and brain. Nutrients such as glucose and amino acids penetrate into the brain via transporters, whereas uptake of larger molecules, including insulin and transferrin, occurs via receptor-mediated endocytosis [5, 6]. Among the factors controlling the passive entry of drugs into the CNS, lipid solubility is the predominant element because of the lipidic nature of cell membranes [7]. The overall hydrophilic/lipophilic balance of a molecule appears to be a better predictor of BBB permeability than the octanol/buffer partition coefficient. Molecular size, to which the rate of solute diffusion is inversely related, also appears to be relevant for hydrophilic compounds, but does not significantly influence the BBB permeability of lipophilic compounds. Aside from passive diffusion through lipid membranes, the binding of molecules to plasma proteins, ionization at physiological pH (pKa), affinity and capacity of transport systems and potential BBB/cerebral metabolism are also important for entry into the brain. There are also an increasing number of studies showing that the activity of the Blood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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efflux transporter P-glycoprotein (P-gp) at the BBB prevents significant accumulation of many hydrophobic molecules or drugs in the CNS [8, 9].

19.2 The Multidrug Transporter P-Glycoprotein

Almost two decades ago, Juliano and Ling showed that “Drug-resistant Chinese hamster ovary cell membranes possess a carbohydrate-containing component of 170 000 daltons apparent molecular weight which is not observed in wild type” [10]. They were the first to demonstrate that overexpression of a glycoprotein they called P-glycoprotein (P for permeability) was responsible for pleotropic resistance of tumor cells against a wide variety of chemotherapeutic agents. Thus, the development of simultaneous resistance to multiple drugs (MDR) that occurs after selection for resistance to a single agent mimics the MDR of human tumors treated with chemotherapy. Therefore the ATP-dependent efflux pump of anticancer drugs, P-gp, is one of the main causes of failure in chemotherapy. P-gp is a member of the ATP-binding cassette (ABC) group of transporters which represent the largest family of transmembrane proteins. They are found in all prokaryotic and eukaryotic cells. The vast majority of ABC proteins are active transporters requiring ATP hydrolysis to provide a driving force to translocate substrates against a concentration gradient across cell membranes. The first ABC transporter was sequenced more than 20 years ago [11]. Proteins are classified as ABC transporters based on the sequence and organization of their ATP-binding domains, also known as nucleotide-binding folds (NBFs). The NBFs contain characteristic motifs (Walker A and B motifs), which are found in all ATP-binding proteins [12]. Some 48 members of the ABC superfamily have been described in humans [13, 14]. They are divided into seven families (ABC A–G). The standard nomenclature, developed by the Human Genome Organization, is available at http:// www.gene.ucl.ac.uk/nomenclature/genefamily/abc. Three of the ABC subfamilies (C, B, G) contain transporters known to express significant transport activity in the BBB and in the blood-cerebrospinal fluid barriers (BCSFB): P-gp (ABCB1), MRP (ABCC1, 4, 5, 6) and BCRP (ABCG2). These play a pivotal role in the brain barrier by functioning as active efflux pumps.

19.2.1 P-gp Isoforms

Among the three efflux protein families known to have activity at the BBB, Pgp is one of the important ones identified at the BBB. It has been proposed that P-gp plays an important role at the BBB in limiting the entry of many drugs into the brain [15, 16]. The expression of P-gp in human BBB endothelial cells was first described by Cordon-Cardo et al. and Thiebaut et al. in 1989 [17, 18]. P-gp is encoded by the MDR1 gene in humans and by mdr1a and mdr1b in ro-


dents and is associated with the MDR phenotype. P-gp encoded by MDR2 in humans or by mdr2 in rodents does not play an important role in the transport of drugs [19]. Mice genetically deficient in the mdr1b gene or in both mdr1a and mdr1b genes have normal viability. However, they show an accumulation of various drugs in the brain and other tissues, indicating that P-gp may act as a guardian by preventing the passage and accumulation of many drugs in the brain [9, 20, 21]. Moreover, it was shown that P-gp could limit the access of naturally occurring molecules, such as the glucocorticoid cortisol, to the mouse and human brain, particularly to the hippocampal area [22, 23].

19.2.2 Structure

The P-gp human product of MDR1 has 1280 residues whereas the mouse proteins mdr or mdr3 have 1276 residues. A model for the structure of P-gp has been proposed based on the amino acid sequences (Fig. 19.1). In this model, two homologous halves (43% homology between the cDNA sequences) comprise each membrane transporter. Each half contains six alpha-helix transmembrane-spanning segments (TMs), for a total of 12 TMs. One cytoplasmic domain containing an ATP-binding site is also found on each half of the molecule. Phosphorylation sites have been identified in the linker region between the two halves of MDR1 in human [24] and two to four glycosylation sites have been described in the first extracellular loop between TM1 and TM2. Thus, the variability in the measured molecular weight of P-gp (150–180 kDa) is presumably due to different levels of P-gp glycosylation between species and tissues [24–26]. Using different biochemical approaches, various oligomeric states have been reported for P-gp, depending on the tissue and cells employed [27, 28]. Electron microscopy and computer models proposed that association between the TMs of P-gp leads to the formation of a pore with a funnel-shape across the membrane [29]. In this 3-D model, the protein viewed from the extracellular side has a diameter of about 10 nm surrounding a central pore of 5 nm diameter. The opening of this pore is narrowed inside the membrane by the nucleotide-binding domain of the protein. A rearrangement of the human P-gp TMs in the presence of different substrates or as a result of ATP hydrolysis has also been proposed [30–33]. From their studies, Loo and Clarke proposed a number of amino acid residues that could be involved in the interactions of, and thus the binding domains for, various P-gp substrates. Data obtained with transport measurements of fluorescent substrates and photoaffinity labeling studies support the existence of different binding sites for P-gp substrates, inhibitors or modulators [34–36]. Recently, a crystallographic structure of P-gp has been reported which demonstrates that, upon binding ATP, the TMs undergo a reorganization in compact domains [37]. In addition, models representing two different functional states of P-gp (nucleotide-free, nucleotide-bound) have been proposed for the conformational rearrangement of TMs [38]. Despite the voluminous data on

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Fig. 19.1 Schematic representation of P-gp. The P-gp secondary structure, embedded in the cell membrane, is presented. P-gp possesses 12 transmembrane domains; and N-glycosylation, phosphorylation and ATPbinding sites are also indicated. The most common nonsynonymous polymorphisms, which induce encoded amino acid changes, and synonymous polymorphisms, which cause a silent mutation, are also shown.

P-gp interactions with its ligands, the direct structure-activity relationships (SAR) of P-gp remains to be clearly established. Better development of the SAR, which would increase the understanding of the pharmacological and physiological significance of P-gp, could eventually help in the prediction of drug entry into the brain through the BBB.


19.2.3 P-gp Substrates

The first molecules identified as P-gp substrates were generally from natural sources, either plants or microorganisms. Many drugs are transported by P-gp and their accumulation in the brain is limited (Table 19.1). Vinca alkaloids, epipodophyllotoxins, anthracyclines and taxanes are among the anticancer agents known to be transported by P-gp [39–41]. Increasingly, molecules other than anticancer agents have been identified as P-gp substrates. For example, P-gp transTable 19.1 Substrates of P-gp. Compound

Ref.

Anticancer agents Actinomycin D Anthracyclines Colchicine Daunorubicin Dexamethasone Docetaxel Doxorubicin Etoposide Mitomycin C Paclitaxel (taxol) Tamoxifen Vinblastine Vincristine

178 181 183 178 82, 188, 189 190 192, 193 194 178 178 199–201 178, 203 178

Immunosuppressive agents Cyclosporine A Rapamycin Sirolimus Tacrolimus

57, 204, 205 207, 208 211 212

Others Opioids (morphine) Erythromycin (antibiotic) Okadaic acid Steroids Aldosterone Cortisol Corticosterone Glucocorticoids Progesterone Sphingomyelin Lovastatin (lipid-lowering agent)

219, 220 222, 223 225 44, 227 44, 228 23, 44 229 44, 230 44 231 226

Compound

Ref.

Ca2+ channel blockers Diltiazem Mibefradil Verapamil

179, 180 182 184–187

Fluorescent dyes Rhodamine 123 Hoechst 33342 Calcein-AM Tetracycline Tetraphenylphosphonium Ramosetron

191 35, 36 195, 196 197 198 202

HIV protease inhibitors Amprenavir Indinavir Saquinavir Ritonavir

206 209, 210 209 213

Bioactive peptides Adrenorphin Endomorphin 1 and 2 Somatostatin b-Amyloid

48 48 49, 218 87

Cardiac drugs Digoxin Quinidine Digitoxin Substance P

214, 215 178, 216 217 49

Toxic peptides Valinomycin Gramicidin D

221 224

Cytokines Interferon-c Interleukin-2 and -4

45, 46 47

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ports cardiac drugs, Ca2+ channel blockers, HIV protease inhibitors, immunosuppressive agents, fluorescent dyes and cyclic and linear peptides [40–43]. In addition to xenobiotics, various endogenous substrates for P-gp have been identified in normal tissues, including several steroids such as cortisol, corticosterone, progesterone and aldosterone [23, 44]. Also, cytokines (IL-2, IL-4, IFN-c) and bilirubin have also been shown to be transported by P-gp [45–47]. Recently, the transporter has been shown to have high affinity for endogenous bioactive peptides, such as adrenorphin, endomorphin 1 and 2, somatostatin and substance P [48, 49]. Studies have shown that molecular weight (MW), surface area, aromaticity, amphiphilicity, proton basicity and H-bond accepting are important in determining P-gp substrate specificity [50–52]. Recently, it was proposed that P-gp substrate specificity could be approximated by three rules obtained from the MW, the H-accepting capacity (given by the Abraham’s b coefficient) and from the ionization which is represented by the acid and base pKa values of compounds. Thus, compounds with an Abraham’s b coefficient ³ 8 (approximately the total number of N and O atoms), MW > 400, and pKa > 4 are likely to be Pgp substrates, whereas compounds with (N + O) £ 4, MW < 400 and pKa < 8 are likely to be non-substrates [53]. The application of this model could be useful in absorption, distribution, metabolism and excretion (ADME) profiling of new drugs. However, since P-gp possesses multiple binding sites and complex mechanisms for substrate recognition and transport, SAR models remain difficult to develop. The prediction of P-gp substrate specificity is influenced by several factors, including the types of assays used, the confusion between P-gp substrates and inhibitors and the binding possibilities with other targets such as cytochrome P450 3A4 [43, 53]. Reversal agents are molecules that restore sensitivity to anticancer agents in drug-resistant cancer cells by inhibiting the transport activity of P-gp. Three generations of these compounds have been used so far (Table 19.2). For example, calcium channel blockers, calmodulin antagonists, quinolins, steroids, immunosuppressive agents, antibiotics and detergents are reversal agents known in the first generation [54–56]. However, most of these agents produce significant toxicities when used at concentrations sufficient to inhibit P-gp. Several of these compounds are themselves substrates for P-gp and for other transporters. Among them, cyclosporin A (CsA) and verapamil were most often employed but cannot be used safely for MDR reversal at the dosage required. This led to the development of second-generation P-gp modulators, such as SDZ PSC 833 (valspodar), a CsA analogue [57]. Most of these agents have the same pharmacological properties as the original molecules but with less toxicity. In spite of their efficiency, many characteristics limit their clinical usefulness. It has been demonstrated that these compounds can significantly inhibit the metabolism and excretion of cytotoxic agents [58]. The high toxicity associated with this side-effect requires a reduction of chemotherapeutic doses in clinical studies [58]. Also, several second-generation P-gp modulators are themselves often substrates for cytochrome P450 3A4 enzyme or other transporter proteins such as

19.2 The Multidrug Transporter P-Glycoprotein Table 19.2 Modulators of P-gp properties. Modulator

Ref.

Medical use/analogy/type a)

First-generation compounds Cyclosporin A Nifedipine Progesterone Quinidine Quinine Tamoxifen Verapamil

57, 204, 205 232 233 178, 216 234, 235 199, 200 184, 185

Immunosuppressive Calcium channel blocker Progestative Antiarrhythmic Antimalarial Antioestrogen Calcium channel blocker

Second-generation compounds Valspodar (PSC833) Cinchonine Dexniguldipine Dexverapamil

28, 236 234 237 238, 239

Cyclosporin A Quinine Nifedipine Verapamil

Third-generation compounds Tariquidar (XR9576) Zosuquidar (LY335979) ONT-093 Tariquidar (XR9576) Biricodar (VX710) Elacridar (GF120918/GG918)

240 241, 242 243 244 60, 245 246

Anthranilamide Difluorocyclopropyldibenzosuberane Substituted diarylimidazole Anthranilic acid derivative Piperidine carboxylate Acridone carboxamide

Natural compounds Curcumin Ginseng Piperine Catechins from green tea Silymarin from milk thistle Garlic

63 85, 247, 248 249, 250 66, 251 252 253, 254

Polyphenol Ginsenosides Alkaloid (black pepper) Polyphenols Flavonoids Organosulfur compounds

a)

First-generation compounds: medical use. Second-generation compounds: analogy to first generation compound. Third-generation/natural compounds: type or chemical structure.

MRP1 [59, 60]. These compounds are in competition with the cytotoxic agent for transport by the pump, giving an unpredictable pharmacokinetic interaction. After disappointing results, a third generation of reversal agents was developed. These molecules aim to specifically inhibit P-gp function. These agents do not affect cytochrome P450 3A4 and were generally developed using SARs and combinatorial chemistry. Because they are noncompetitive inhibitors of the P-gp transporter, the use of this third generation of P-gp modulators permits a reduction in the dosage of chemotherapeutic agents. The clinical efficacy of these reversal agents remains to be established, not only with regard to overcoming tumor resistance against chemotherapy, but also for other factors such as bypassing P-gp in the BBB. In recent years, several

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new approaches have been developed to reduce and inhibit MDR1/P-gp expression in cells. Among them, the use of monoclonal antibodies or immunotoxins against P-gp, antisense oligonucleotides and small interfering RNAs (siRNAs) has been investigated [61, 62]. Furthermore, natural products from dietary intake, such as curcumin ginsenosides and piperine, have been identified as inhibitors of P-gp and several flavonoids, such as quercetin and naringenin, are reported to modulate P-gp activity (Table 19.2) [63–65]. Moreover, we have also demonstrated that epigallocatechin gallate, the major polyphenol present in green tea, inhibits P-gp activity [66]. At the same time, much effort has gone into investigating and identifying new natural compounds that inhibit P-gp, reverse the MDR phenotype and sensitize cancer cells to conventional chemotherapy without toxicological effects. However, other studies are necessary to understand the mechanisms involved in P-gp modulation by these natural products and to explore their potential in chemoprevention.

19.3 Localization and Transport Activity of P-gp in the CNS 19.3.1 Normal Brain

P-gp is found in many normal tissues with excretory function, including liver, kidney and small intestine [67, 68], and at blood-tissue barriers such as the BBB, blood-testis barrier and placenta [69]. As a result of its anatomical localization, P-gp is one of the most important transporters for drug disposition in the organism. It limits drug entry into the body after oral drug administration (enterocyte luminal membrane), it promotes drug elimination into bile and urine (hepatocyte canalicular membrane, kidney proximal tubule luminal cell membrane) and it limits drug penetration into sensitive tissues (brain, testis, fetal circulation). The expression of P-gp in human BBB endothelial cells has been described in many studies, performed in various species (human, rat, mouse, cow, pig) [70– 72] and indicates that the major site of BBB P-gp expression is at the luminal membrane of capillary endothelial cells (Fig. 19.2 A, left panel). Several reports have shown that P-gp could be also present in the brain parenchyma. For example, in vitro P-gp expression and activity have been demonstrated in primary astrocyte rat brain cultures [73, 74] and in microglia [75]. In vivo, a recent study examined Pgp distribution, using confocal microscopy on rat brain sections and indicated that this transporter was preferentially expressed in the endothelial component but was also present in astroglial cells [76]. Another team observed that the P-gp pattern of expression in human and primate brain was the same as that seen for the astrocyte marker GFAP [77, 78]. Based on these immunofluorescent studies, a model of MDR in brain was proposed where P-gp is localized on astrocyte foot processes at the antiluminal side of the human BBB. More recently, this group pub-

19.3 Localization and Transport Activity of P-gp in the CNS

Fig. 19.2 P-gp expression at the BBB. (A) Left panel: Detection of P-gp in homogenates and in endothelial cells (EC) isolated from brain using magnetic cellsorting beads. Right panel: Proteins from whole membranes, brain capillaries and endothelial luminal membranes were separated by SDS-PAGE and immunodetected with mAb P-gp (C219) antibody.

(B) Schematic view of P-gp at the BBB. EC are sealed by continuous tight junctions and surrounded by a basal lamina. Pericytes are present at the periphery of vessels. Astrocyte foot processes are in close contact. P-gp is present in the luminal membranes of the brain vascular endothelium and impedes brain penetration by lipophilic substances.

lished results where P-gp expression was found both in astrocytes and in endothelial cells of healthy primate brain [79]. In our laboratory, a study indicated that P-gp was strongly enriched in the positive endothelial cell fraction from brain and was absent from the negative fraction in which the glial fibrillary acidic protein (GFAP), an astrocyte marker, was present [80]. It was also shown by RT-PCR analysis that the mdr1a gene was preferentially expressed in this enriched EC fraction from the brain. At the subcellular level, our findings demonstrated that the P-gp

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was localized in isolated luminal membranes from the brain vascular endothelium in rat [81] (Fig. 19.2 A, right panel). Overall, data obtained for most studies suggest that P-gp expressed in the capillary endothelial cells of the BBB restricts the CNS accumulation of many drugs, including chemotherapeutic agents (Fig. 19.2 B). This protective action of P-gp has been demonstrated using mdra knockout mice [82]. In addition to the expression of P-gp at the BBB, there have been a few reports of the expression and functional activity of P-gp in the choroid plexus [76, 83, 84]. P-gp was localized at the subapical side of choroid plexus epithelia and vectorial transport experiments performed on cultured rat choroid plexus showed an apically directed efflux function for P-gp, suggesting a role in preventing the export of certain substances out of the CSF, as opposed to its action at the BBB. More studies are needed to understand and characterize the role of P-gp at the choroid plexus.

19.3.2 Brain Diseases

The expression and activity of P-gp in the CNS plays an important role in the disposition and efficacy of pharmacological agents for brain diseases, such as brain tumors, epilepsy or HIV-associated dementia [85, 86]. In addition, P-gp also seems to play a key role in the etiology and pathogenesis of certain neurological disorders, such as Alzheimer’s and Parkinson’s diseases [87, 88]. In brain tumors, progress in clinical treatment has been slow and one of the major problems impeding treatment of these tumors is their weak response to anticancer drugs. In fact, brain tumors are known to develop MDR quite rapidly. Furthermore, gliomas are characterized by their infiltrative pattern of growth and it is likely that the blood-brain area in the tumoral periphery, which often escapes surgical intervention, possesses a totally functional BBB. The low response to chemotherapy may also depend on tumor blood flow, the integrity of the blood-tumor barrier and an inherent or acquired MDR phenotype in cancer cells [89]. As P-gp plays a major role in the defense of the organism against xenobiotics at the BBB [9], the determination of P-gp levels in brain tumors and peritumoral tissue is crucial for evaluating the long-term efficacy of chemotherapy. P-gp has been detected in endothelial cells from newly formed microvessels of gliomas [90–92], suggesting that, despite the leaky nature of the vasculature of gliomas, angiogenic vessels have maintained some of the restrictive capacities of the BBB.

19.3.2.1 Malignant Brain Tumors We have reported that the P-gp expression levels detected by Western blot in various human malignant brain tumors (low-, high-grade gliomas) are similar to the levels of P-gp expression found in normal brain [93]. This is in agreement with previous studies which reported the presence of P-gp in resistant


and partially chemosensitive glioblastomas by immunohistochemistry, using the monoclonal antibody C219 [94–96]. These results suggest that the poor response of brain tumors to many anticancer drugs may be related to the presence of this efflux transporter in cell populations of the primary brain tumors and that P-gp may be considered as a negative factor when predicting the outcome for patients with brain tumors. These findings also suggest that P-gp expression is maintained in both low- and high-grade gliomas. Moreover, the widespread expression of P-gp in these tumors may reflect an intrinsic resistance to anticancer drugs. Previous immunohistochemical analyses showed that most gliomas and, more specifically, endothelial cells within the gliomas, stained positively for MDR1 P-gp [91, 92]. These studies support the concept that clinical drug resistance may be caused by P-gp expression, not only in cancer cells but also in the capillary endothelial cells of brain tumors. The role of the BBB in the low efficacy of chemotherapy is still unclear. Alterations in the brain capillary ultrastructure have been described, leading to an increase in the microvascular permeability in gliomas. In contrast, it has been reported that the neovasculature of even high-grade tumors preserves partial BBB permeability properties at the cellular level [97] and that the BBB at the tumor periphery is still intact. In addition, a study indicated that P-gp, one of the best phenotypic markers of the BBB, is expressed at the same levels in all primary tumors as in normal brain, indicating that brain tumors retain an important characteristic of the BBB which restricts the brain uptake of chemotherapeutic agents. Thus the BBB, especially at the edge of tumors, remains a formidable obstacle for drug distribution to brain regions that have been infiltrated by neoplastic cells [98].

19.3.2.2 Brain Metastases Brain metastases occur in 20–40% of cancer patients and the estimated incidence in the United States is 170 000 new cases per year [99]. Lung cancer (9.7– 64.0%), breast cancer (2–25%) and melanoma (4–20%) are the most common primary sources of metastases to the CNS [100]. Strikingly, we found that brain metastases from melanomas and lung adenocarcinomas exhibit only 5% and 40%, respectively, of the P-gp levels found in normal brain [93]. Metastatic malignant melanomas are recognized for their poor response to chemotherapy, whereas some effects of chemotherapy have been observed for lung adenocarcinomas [101]. The low expression of P-gp in these brain metastases suggests that MDR mechanisms other than P-gp could be responsible for their poor response to chemotherapy. The lack of P-gp expression in primary lung tumors and corresponding brain metastases also indicates that these brain metastases do not acquire the levels of P-gp expression found in normal brain tissue.

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19.3.3 Expression of Other ABC Transporters at the BBB

It has been reported that efflux transporters other than P-gp are also expressed in brain capillaries. For instance, members of the MDR-associated protein (MRP) family have been detected at the BBB. In humans, seven MRP homologues have been identified [102]. All members of the MRP family are distributed throughout most human tissues [103]. MRP1, which was first described in 1992, was immunodetected by Western blots in human and rat choroid plexus, but the presence of MRP1 in the EC of brain capillaries remains controversial [104]. In animal models, Western blot and RT-PCR analysis suggest that MRP1 is expressed in isolated rat brain capillaries, primary cultured rat, pig and cow BCECs and immortalized rodent BCECs [105–107]. However, in isolated human brain capillaries, no expression of MRP1 was observed by immunohistochemistry [108]. The canalicular multispecific organic anion transporter (cMOAT or MRP2) was principally detected in hepatocytes, intestine and kidney but was not detected in endothelial cells of rat brain capillaries by Western blot [109]. Recently, MRP1, -4, -5 and -6 were shown to be expressed in primary BCECs by RT-PCR analysis as well as in a capillary-enriched brain extract [110]. In addition, MRP mRNA levels appeared to be closely associated with resistance to etoposide, adriamycin and vincristine in human glioma cell lines derived from patients [111]. Recently, levels of MDR1 and MRP1– MRP4 mRNA were compared between normal brain tissue and malignant gliomas [112]. The expression of both MDR1 and MRP2 were similar in normal brain and tumors, whereas MRP1 and MRP3 expression increased with tumor grade. Therefore, some of the MRPs may also confer intrinsic MDR activity in human gliomas or in metastatic brain tumors.

19.3.4 Subcellular Localization of P-gp

P-gp was also found in a specialized microdomain of plasma membranes called caveolae. This P-gp expressed in caveolae was first identified in multidrug-resistant cells [113–115], where it appears to play an important role in drug resistance development [116, 117]. Caveolae are flask-shaped plasma membrane invaginations involved in many cellular events such as transcytosis, endocytosis, cholesterol transport and signal transduction [118]. A family of proteins called caveolins comprise the structural component of caveolae. Caveolin-1 and caveolin-2 are primarily expressed in adipocytes, endothelial cells, smooth muscle cells and type I pneumocytes [119], whereas caveolin-3 is expressed in muscle and glial cells [120, 121]. Caveolin-1 possesses two isoforms (a, b) whereas three isoforms (a, b, c) were reported for caveolin-2. MDR is a multifactorial process and recently an upregulation of caveolae and caveolar constituents, such as caveolin-1, -2 and glucosylceramide, was observed in different MDR cancer cells compared to their drug-sensitive counterparts [113, 114, 122, 123].


Localization of P-gp in caveolae has been also shown in the brain by different means. First, using a detergent-free method for caveolae isolation, our group showed the enrichment of P-gp, caveolin-1 and cholesterol in the low-density microdomains of human isolated brain capillaries and endothelial cells of an in vitro BBB model [115, 124]. Second, immunocytochemical analysis demonstrated the presence of P-gp in plasmalemmal vesicles of rat brain capillaries and in an immortalized rat brain endothelial cell line, RBE4 [125]. Then, Virgintino et al. [126] showed, by microscopy, that a large proportion of P-gp and caveolin-1 colocalize in the luminal compartment of the endothelial cells in human microvessels of the cerebral cortex. Besides endothelial cells, immunocytochemical analysis shows that, in astrocytes, a portion of P-gp is localized in caveolae [74] and colocalized with caveolin-1 [79]. In addition to the colocalization of some P-gp and caveolins in caveolae, our coimmunoprecipitation studies indicated that a population of P-gp molecules interacted with caveolin-1 in endothelial cells of the BBB [115, 124]. This coimmunoprecipitation was also reported in MDR cells [115, 127] and in astrocytes [74]. Similar to caveolin-1, caveolin-2 interacts with P-gp; and these three proteins form a high molecular mass complex at the BBB [124]. Oligomeric forms of P-gp have been observed in MDR cells and brain capillaries [28, 128] and recent data suggest that P-gp oligomerizes through indirect interactions [129]. The involvement of caveolins in P-gp oligomerization remains to be investigated, as well as the possibility of other proteins interacting with P-gp, like actin, ezrin, radixin, moesin, calnexin, Hsp70 and Hsp90 beta [130–132]. P-gp contains in its N-terminal portion a consensus caveolin-binding motif present in many proteins known to bind the scaffolding domain of caveolin-1 (Fig. 19.1). Three related caveolin-binding motifs are known (UXUXXXXU, UXUXXXXUXXU, UXUXXXXUXXU, where U is a phenylalanine, tyrosine or tryptophan residue and X is any amino acid residue) [133]. The scaffolding domain of caveolin-1 regulates signaling molecules localized in caveolae such as eNOS, protein kinase C, insulin receptor, EGF and VEGF receptors [134, 135]. In the case of P-gp, mutation of its caveolin-binding motif decreases the interaction between P-gp and caveolin-1 and increases P-gp transport activity, indicating that caveolin-1 negatively regulates P-gp activity [124]. Moreover, overexpression of caveolin-1 in drug-resistant cells expressing P-gp causes a reduction of P-gp activity and in the cells become drug-sensitive, supporting the hypothesis that caveolin-1 inhibits drug transport by P-gp [127].

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19.4 Polymorphisms of P-gp 19.4.1 MDR1 Polymorphisms at the BBB

In recent years, researchers have started to investigate the molecular mechanisms underlying inter-individual differences in the pharmacological effects of drugs. Genetic variations in drug transporters have received particular interest since they are among the factors determining the pharmacokinetic profile of drugs. Efforts have also been made to identify genetic variations of the human MDR1 (ABCB1). Single nucleotide polymorphisms (SNPs) result in a single nucleotide substitution and possibly a change in the encoded amino acid. More than 40 SNPs and insertion/deletion polymorphisms in the ABCB1 gene have been reported. MDR1 gene SNPs are located in the coding region and in the noncoding region, including the core promoter region and the intron-exon boundaries [136– 138]. The most common allelic combinations of MDR1 SNPs, which encode no amino acid changes, are synonymous polymorphisms at exon 12 (C1236T) and exon 26 (C3435T) and nonsynonymous polymorphisms (encoding amino acid changes) at exon 21 (G2677T). The localization of these major SNPs is shown in the schematic representation of P-gp (Fig. 19.1). Allele frequencies vary widely in MDR1 SNPs, particularly between populations of African descent and other ethnic groups. A large discrepancy is observed in these populations with an average of 18% and 48% frequencies, respectively, for the T allele of the three common allelic combinations of MDR1 SNPs (Table 19.3). Moreover, the segregation observed in the African American population consistently presents different, specific genetic combinations of MDR1 SNPs (haplotype) [136, 138, 139]. Consistent with wild-type allelic frequency in individuals of African origin, epidemiologic studies have observed a lower incidence of ulcerative colitis in Africans as compared with Caucasians [140]. It was suggested that the higher frequency of the wild-type allele (CC) in the African population for exon 26

Table 19.3 Most frequent allelic combinations of MDR1 genetic variations [136–138]. CA = Caucasian; AA = African American; AS = Asian American; ME = Mexican American; PA = Pacific Islander. Exon

12 21 21 26

SNP

C1236T G2677T G2677A C3435T

Allele frequency (%) CA

AA

AS

ME

PA

35–46 42–46 2–10 48–56

21 10 0.5 20–23

68 45 6–22 40–49

45 40 0 50

57 36 36 50

19.4 Polymorphisms of P-gp

(C3435C) SNPs may have resulted in a selective advantage against intestinal tract diseases [141]. There have also been no cases of neurotoxicity reported after treatment with ivermectin for the prevention of onchocerciasis in Africa, even though this drug causes neurotoxicity in animals with low P-gp expression [139, 142]. Thus, it has been demonstrated that some MDR1 polymorphisms have an impact on P-gp expression and function. The introduction of nucleotide changes in highly conserved regions of the MDR1 gene has a major impact on P-gp function and expression, in comparison with a nucleotide substitution introduced in less conserved regions of the gene [40, 143–145].

19.4.2 MDR1 Polymorphism and Brain Pathologies

At the BBB, impairment of P-gp function or altered P-gp expression level has been associated with severe neurotoxic side effects following administration of drugs or xenobiotics [146–148]. The SNPs in exon 26 (C3435TT or CT) genotypes are associated with low P-gp expression in the BBB in comparison to the CC genotypes references. A five-fold increased risk for developing Parkinson’s disease was found in exon 26 (C3435T) heterozygous (T) and homozygous (TT) patients exposed to pesticides [88, 149]. Children with acute lymphoblastic leukemia (ALL) with the C3435TT or CT genotypes demonstrated a better response to chemotherapeutic drugs (e.g. etoposide, vincristine, doxorubicin), thus reducing the risk of CNS relapse [150]. Furthermore, the nonsynonymous G2677T SNPs in exon 21, combined with the synonymous C3435T SNPs in exon 26, increased the neurotoxicity of the immunosuppressive drug tacrolimus in liver transplant patients [151]. Patients with resistance to epileptic drugs have shown a higher frequency of the C3435CC genotype than the C3435TT SNPs. The well known C3435T polymorphism is silent (no amino acid changes) and raises the possibility that the polymorphism is not in itself causal but that different mutational groups (SNPs) forming different haplotypes in a consistent network are possibly the causal events [152]. Thus, linkage disequilibrium of the C3435T SNPs with other SNPs has underscored the importance of understanding haplotypes. Interstudy comparison of the polymorphic effects on P-gp expression and function requires extensive haplotype analyses [136–138, 153, 154]. This will also provide a powerful tool for predicting and optimizing drug therapy, particularly for drugs with narrow therapeutic indices where induction or inhibition of transporter function can have a tremendous impact on drug efficacy and safety [136, 138, 155–157].

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Fig. 19.3 Roles of P-gp at the BBB. P-gp at the BBB could have different physiological roles such as: (1) protection against xenobiotics, (2) secretion of brain endogenous substrates and (3) endothelial secretion.

19.5 Role of P-gp at the BBB

In brain capillaries, P-gp appears to play an important role in preventing many hydrophobic molecules from crossing the BBB and reaching the CNS. However, the exact physiological function of P-gp in the BBB is not completely understood. A growing body of evidence links P-gp to physiological roles distinct from its initially recognized function as a drug efflux system (Figs. 19.3 and 19.4).


Fig. 19.4 Proposed roles of P-gp localized in caveolae. (A) Low-density caveolae-enriched domains were isolated from bovine brain capillary endothelial cells (BBCEC) cocultured with astrocytes, using a carbonate-based fractionation method. Each fraction from a sucrose gradient was separated by SDS-PAGE and immunodetected using antibodies directed against P-gp (mAb C219) and caveolin. (B) Since cholesterol is important for P-gp

activity, the localization of P-gp in enriched cholesterol microdomains (caveolae) contributes to decreasing intracellular drug concentrations by pumping drugs inside caveolar vesicles and increasing their elimination outside the cells. (C) P-gp mediates cholesterol redistribution from the cytosolic leaflet to the exoplasmic leaflet of the plasma membrane. C = Cholesterol; Cav = caveolin-1.

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19.5.1 Protection Against Xenobiotics

Numerous reports provide functional evidence for P-gp-mediated drug efflux at the BBB. The interaction of drugs with P-gp in rat brain capillaries was demonstrated by photoaffinity labeling [158]. The generation of transgenic mice with a disruption of the mdr1a gene provided a pharmacological tool for the study of P-gp function in the BBB [9, 20]. These mice are viable, fertile and do not display obvious phenotypic abnormalities, indicating that this protein is not essential to their vital functions. However, P-gp substrates accumulate in the brains of these mice to a much greater extent than in wild-type animals and they are more sensitive to central neurotoxicity. For example, knockout mice are 50–100 times more sensitive to the neurotoxic effects of the pesticide ivermectin. The accumulation of this drug in brain tissue of mdr1a(–/–) mice was increased 80– 100 times as compared to control mice. Recent application of in situ brain perfusion to wild-type and P-gp-deficient mdr1a(–/–) mice made it possible to assess the influence of P-gp on brain uptake of substrates without the potentially confounding differences in systemic pharmacokinetics upon P-gp distribution [159]. In summary, as indicated by Schinkel in 1999, P-gp appears to be a major efflux transporter at the BBB that acts as a guardian of the CNS by preventing the accumulation of many drugs in the brain [9].

19.5.2 Secretion of Endogenous Brain Substrates and Endothelial Secretion

In addition to its guardian role, P-gp is involved in the excretion of toxic compounds by renal proximal tubules and hepatic canalicular membranes [68, 69] and in the secretion of endogenous molecules from adrenal glands [160]. Thus, P-gp could fulfill a similar function in the BBB and be responsible for the secretion and/or excretion of brain-derived substances or metabolites into the blood (brain secretion). It could also be involved in the secretion of molecules from the endothelium itself (capillary secretion). In this respect, P-gp has been proposed to be involved in the release of neuroactive substances from the brain directly into the systemic blood following intracerebroventricular injection [161]. In addition, it has been demonstrated that b-amyloid (Ab) is transported across the plasma membrane of P-gp-enriched vesicles in an ATP- and P-gp-dependent manner, suggesting that Ab might be an endogeneous substrate for P-gp in brain [87]. Thus, a change in MDR1 function or expression might alter the clearance of Ab from the brain and may contribute to cerebrovascular angiopathy. Since the accumulation of Ab in the brain is a feature of Alzheimer’s disease, the mechanism of Ab transport opens new avenues in the understanding of Alzheimer’s disease.


19.5.3 Caveolar Trafficking

Why P-gp is localized in caveolar microdomains remains to be established. However, several roles can be proposed for P-gp in these membrane microdomains, which are illustrated in Fig. 19.4. As mentioned, a portion of the P-gp localized in the brain capillary endothelial cells was found in caveolae microdomains (Fig. 19.4 A). It has been proposed that the P-gp localized in the caveolae of MDR cells might act to decrease intracellular drug concentrations by pumping drugs inside caveolar vesicles and increasing their elimination (Fig. 19.4 B). Different observations in MDR cells support this role of P-gp in caveolae: increase of caveolae and caveolar constituents in MDR cells compared to their drug-sensitive counterparts [113, 114, 116, 117, 123, 162], localization of P-gp in caveolae in MDR cells [113, 115] and drug sequestration in P-gp-containing cytoplasmic vesicles in MDR cells [163, 164]. Studies have also reported that a portion of the P-gp expressed at the BBB is colocalized with caveolin-1 in caveolae [125, 165]. Other studies have demonstrated that the P-gp expressed at the BBB can also interact with caveolin-1 [115, 124]. Considering the protective role of P-gp at the BBB in preventing the accumulation of many hydrophobic molecules and potentially toxic substances in the brain, modification of caveolae or caveolin levels might affect brain homeostasis. In this regard, a dramatic decrease in caveolin-1 expression has been observed in brain tumor endothelial cells compared to normal brain endothelial cells [166]. Since caveolin-1 inhibits P-gp activity [124, 127], a reduction in caveolin-1 expression could affect drug transport across the BBB and decrease chemotherapy efficiency. In addition, studies from different groups have suggested the involvement of P-gp in lipid transport (Fig. 19.4 C). Studies on P-gp activity, either drug binding or drug transport, in cells where P-gp is localized in caveolae or low-density microdomains show that P-gp is functional in these cholesterol-enriched microdomains [124, 167]. Moreover, studies have shown that cholesterol is important for the activity of P-gp, suggesting that caveolae might provide a favorable environment for its activity [168–170]. Specifically, cholesterol could interact with the substrate binding site of P-gp, suggesting that cholesterol may be transported by MDR1 P-gp (Fig. 19.4 C) [171]. Furthermore, one study has shown that P-gp mediates the ATP-dependent relocation of cholesterol from the cytosolic leaflet to the exoplasmic leaflet of the plasma membrane, suggesting that P-gp might contribute to stabilizing caveolae [172]. It was also reported that caveolin-1 binds cholesterol and mediates its efflux within caveolae via an identified, cytosolic caveolin-1 complex comprising heat-shock protein 56, cyclophilin A and cyclophilin 40, which carries cholesterol to the plasma membrane caveolae [173]. In addition, P-gp in caveolae might contribute towards decreasing the formation of ceramide, which is involved in apoptosis induction [174]. Elevated levels of glucosylceramide, the precursor of ceramide, were observed in MDR cells and appeared to be due to the high activity of glucosylceramide synthase activity. Also,

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it has been shown that sphingomyelin (SM) and the enzyme converting SM into ceramide, called sphingomyelinase, are enriched in caveolae in MDR cells [175, 176]. However, further studies are required to have a better understanding of the role of P-gp in caveolae at the BBB.

19.6 Conclusions

Overall, P-gp plays an important role in brain protection at the BBB. Its expression at the luminal side of endothelial cells in brain capillaries prevents the passage of many agents into the brain [9, 81, 177]. Moreover, many studies have proposed that the capillary EC of brain tumors participates in the resistance associated with P-gp expression, especially at the edge of tumors, where the BBB remains a formidable obstacle for the penetration of anticancer drugs into the brain regions infiltrated by cancer cells [98]. The development of P-gp inhibitors in order to reverse the MDR phenotype has been extensively investigated with generally disappointing results. The current, third-generation inhibitors present high potency and specificity for P-gp. Further studies are required to establish their contribution to potential therapeutic treatment by reversing P-gp-mediated MDR. It was recently reported, in MDR cells, that a portion of the P-gp present in the endothelial cells of the BBB is localized in caveolar microdomains [113, 115]. This particular localization could be useful for understanding the function and regulation of P-gp in drug elimination and transport across the BBB. Finally, recent observations have challenged the notion that P-gp has evolved merely to mediate the efflux of xenobiotics and raised the possibility that P-gp and related transporters might play a fundamental role in regulating cell differentiation, migration, proliferation and survival [174].

Acknowledgments

This work was supported by a grant to R.B. from the Natural Sciences and Engineering Research Council of Canada.

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20 Targeting of Neuropharmaceuticals by Chemical Delivery Systems Nicholas Bodor and Peter Buchwald

20.1 Introduction

Evolution built very efficient ways to isolate and protect the brain, this delicate, but crucial organ that has many vital functions. Unfortunately, the same mechanisms that prevent intrusive blood-borne substances from accessing the brain also prevent therapeutic chemicals from doing the same. Many pharmaceuticals are ineffective in the treatment of cerebral diseases because we cannot efficiently deliver and sustain them within the brain, a considerable problem considering the prevalence of brain diseases. More than 15% of the United States adult population (> 18 years) is affected by some form of mental disorder: ~ 5% affective disorders, ~ 7% anxiety disorders, ~ 4% substance abuse/dependence, and ~ 1% severe cognitive impairment (1988 data) [1]. Despite almost twice as many people suffering from central nervous system (CNS) disorders than from cardiovascular diseases, the global market for CNS drugs is only about half of that for cardiovascular drugs (in the USA) [2, 3]. In light of these numbers, it is obvious that any drug delivery method that can provide brain delivery or eventually preferential brain delivery (i.e., brain targeting) is of great interest. Here, we present a review of the field of brain-targeted chemical delivery systems (CDSs), a rational drug design approach that exploits sequential metabolism not only to deliver, but also to target drugs to their site of action. To illustrate the complexity of the problems that have to be overcome for successful brain targeting, a brief overview of the blood-brain barrier (BBB) and other issues related to brain targeting is also included.


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20.2 The Blood-Brain Barrier 20.2.1 Structural Aspects

The cerebral blood compartment does not have free diffusional communication with the interstitium of the brain: unlike most other organs, the brain is tightly segregated from the circulating blood by a unique membranous barrier, the BBB [4–13]. Capillaries in the brain and spinal cord are lined with a layer of special endothelial cells that lack fenestrations and are sealed with tight junctions; and therefore, these capillaries lack the small pores that allow the rapid movement of solutes from circulation into other organs (Figs. 20.1 and 20.2). These endothelial cells, together with perivascular elements such as astrocytes and pericytes, constitute the BBB. Neuronal endings may also directly innervate the endothelium [14, 15]. A barrier seems to be present only in animals with a complex level of higher neural function, and, at least based on qualitative observations, “higher” functions of neural tissue seem to be protected by a tighter barrier [16]. The presence of a barrier only in invertebrate groups capable of complex CNS functions might also indicate that a barrier is needed when the level of integrative activity in the nervous tissue reaches a critically sophisticated level [16]. Precise synaptic signaling might indeed require a finely regulated cerebral ionic microenvironment. Nevertheless, certain areas within the brain (circumventricular organs) are “outside the barrier” in the sense that they lack a tight epithelium, but appreciable leakage across these sites is unlikely as they account for only about 0.0002 of the surface area of the blood-brain exchange [16, 17]. Intercellular clefts, pinocytosis, and fenestrae are virtually nonexistent in brain capillaries, and hence, diffusion has to take place transcellularly (Fig. 20.2). Consequently, only the lipid-soluble solutes that can freely diffuse through the capillary endothelial membrane may passively cross the BBB. In general capillaries, such an exchange is overshadowed by other nonspecific exchanges. The brain is an organ of high metabolic rate, and therefore, of high blood flow. Cerebral blood flow takes up a considerable portion of the total cardiac output, and the brain’s extensive network of capillaries could bring any compound within easy reach of neurons. However, despite an estimated total length of 650 km and total surface area of 12–20 m2 of capillaries covered by only ~ 1 ml of total capillary endothelial cell volume in an average human brain of 1.3 kg [15], the BBB is very efficient and makes the brain practically inaccessible for lipid-insoluble compounds, such as polar molecules and small ions. Microvessels make up an estimated 95% of the total surface area of the BBB, and they represent the principal route by which chemicals enter the brain [18]. Capillaries in the brain are separated by only about 40– 70 lm, and hence, the chemicals that can pass through the BBB can rapidly reach any brain tissue by diffusion. Vessels in the brain seem to have somewhat smaller diameters and thinner walls than vessels in other organs (Fig. 20.1) [19]. Also, the

20.2 The Blood-Brain Barrier

General capillary (ciliary body)

Brain capillary

Fig. 20.1 Capillary profiles from ciliary body and brain gray matter in rat, shown at slightly different scales. L = Lumen; m = mitochondrion; Nu = endothelial nucleus; P = pericyte. (Reprinted from [19] with permission of Wiley-Liss, Wiley Publishing Inc.).

Fig. 20.2 Schematic comparison of general and brain capillaries.

mitochondrial density in brain microvessels seems to be somewhat higher than in other capillaries, not because more numerous or larger mitochondria are present, but because the small dimensions of the brain microvessels and the consequently smaller cytoplasmic area make the mitochondrial density appear higher [19]. The transendothelial electrical resistance, a measure of ionic permeability through

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junctions, has been estimated to be 1000–5000 X cm–2 for brain microvessels, in contrast to values of about 10 X cm–2 for most noncerebral capillaries [17]. The BBB is disrupted following acute stroke and trauma (see Chapter 25), and it is often assumed that in such cases all compounds can be delivered by an intravascular approach. However, the situation is more complex, as openings are usually localized and biphasic, and the BBB still impedes the delivery of therapeutics [17]. Brain tumors may also disrupt the BBB, but these are also local and nonhomogeneous disruptions [20]. Brain tumors probably contain capillaries with intact BBB as well as capillaries with increased permeability. Like many other things in modern medicinal chemistry, the concept of a barrier between blood and brain can be traced back to the work of Paul Ehrlich, who noticed around 1885 that i.v. injection of dyes such as trypan blue in laboratory animals colored all tissues except the brain [21]. He originally thought that the brain has a lower affinity for these dyes than other tissues, but in 1913 Edwin Goldmann, one of his students, showed that injection of trypan blue directly into the cerebrospinal fluid of rabbits and dogs colors the brain without staining other organs [10]. This was a clear indication that the CNS is separated from the blood by some kind of a barrier; but, concerning the exact nature and localization of this barrier, a long period of uncertainty followed. For a long while, it was thought that astrocytes, members of the largest class of brain cells – the glial cells, must constitute the BBB because brain capillaries are almost completely surrounded by astrocyte foot processes. This later proved false, but the exact role of astrocytes is still not completely clarified, and most likely, they are involved in stimulating the development of barrier properties in the capillary endothelia [22]. Spatz suggested the capillary endothelium as an essential structure of the barrier in 1933 [23], and later, Crone also argued in favor of a peculiar tightness of the cerebral endothelium [24]. Nevertheless, only Reese and Karnovsky’s 1967 experiment with horseradish peroxidase [25], followed by that of Brightman and Reese [26], provided a convincing proof for this idea.

20.2.2 Enzymatic and Transporter-Related Aspects

The BBB also has an additional, enzymatic aspect. Solutes crossing the cell membrane are exposed to degrading enzymes present in large numbers inside the endothelial cells that contain large densities of mitochondria, metabolically highly active organelles. Enzymes and receptors found in the BBB include, among others: adenylate cyclase, guanylate cyclase, Na+–K+ ATPase, alkaline phosphatase, catechol O-methyl transferase (COMT), monoamine oxidase (MAO), GABA transaminase, and DOPA decarboxylase [8, 27–30]. BBB enzymes also recognize and rapidly degrade most peptides, including naturally occurring neuropeptides [31, 32]. Active transporters also play an important BBB-role. While pointing to lipid solubility as an important factor determining access to the brain, in 1946 Krogh had already raised the possibility of some sort of active transport processes in

20.3 Brain-Targeted Drug Delivery

the endothelium of the vessels in the CNS [33]. Today, it is well known that a few special solutes have access to specific, catalyzed transport, for example, the glucose transporter ensures that glucose, an essential nutrient, reaches the brain despite its low lipid solubility [34, 35]. Carriers are facilitative in nature and have been identified for hexoses, amino acids, peptides, monocarboxylic acids, organic cations, nucleosides and nucleoside bases, choline, and other biologically important molecules [36, 37]. These protein macromolecular systems are often characterized by saturability and molecular selectivity. Because this avenue provides for the movement of selected molecules, a number of attempts were made to exploit it for drug delivery purposes by coupling the therapeutic agent that has to be delivered to one of these molecules. More recently, active efflux pumps have also been identified; and this recognition has considerably impacted CNS drug development [38]. The existence of probenecid-sensitive, active pumps for organic anions, such as b-lactam drugs or valproic acid, has been suspected for a while [39], and there is now considerable evidence for their existence [40, 41]. According to current knowledge [30, 38], three classes of transporters are implicated in the efflux of drugs from the brain: (1) monocarboxylic acid transporters (MCT), (2) organic ion transporters [organic anion transporters (OAT), organic cation transporters (OCT), organic anion transporter proteins (oatp), and organic cation/carnitine transporters (OCTN)], and (3) multidrug resistance (MDR) transporters [P-glycoprotein (Pgp), the prototypical MDR transporter, and MDR-associated protein (MRP)]. P-gp garnered considerable attention within the brain delivery field, as sufficient hydrophobicity is usually a target for CNS drugs, and classic P-gp substrates tend to be hydrophobic, amphipathic molecules (usually with a planar ring system) [42]. P-gp was first identified in 1976 as playing a role in cellular permeability (hence, P stands for permeability) [43], and it is a membrane protein belonging to the ATP-binding cassette (ABC) transport protein family, but contrary to most other members of this family, it appears to have wide substrate specificity. P-gp is a possible source of peculiarities in drug pharmacokinetics, including dose-dependent absorption, limited oral bioavailability, drug-drug interactions, intestinal secretion, and limited BBB permeability, and thus, it can considerably impact the therapeutic efficacy of its substrates [44]. The involvement of P-gp in active BBB transport has been confirmed by many studies [12, 45–51]. It seems that substrates, or at least some of them, are pumped out from the membrane itself [52].


Considering all these facts, it is not surprising that practically all drugs currently used for disorders that require CNS-delivery are lipid-soluble and can readily cross the BBB following oral administration. The delivery of other therapeutics to the brain requires some strategy to overcome the BBB, and therefore,

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general brain delivery approaches are of obvious therapeutic interest; and many related aspects have been reviewed [2, 17, 20, 32, 53–56]. Delivery of compounds such as neuropeptides or oligonucleotides is further complicated by their metabolic lability [32]. From a drug development perspective, one can aim either to modify existing drugs to increase their BBB penetration or to develop new chemical entities (NCEs) that already possess the desired permeability properties [57]. The spectrum of approaches intended to deliver potential therapeutic agents to the brain includes intracerebral delivery, intracerebroventricular delivery, intranasal delivery, BBB disruption, nanoparticles, receptor-mediated transport (vector-mediated transport or “chimeric” peptides), cell-penetrating peptides, prodrugs, and chemical delivery systems, which have been reviewed recently [56].

20.3.1 Lipophilicity and Its Role in CNS Entry

There has been a more or less continuous interest in defining lipophilicity/hydrophobicity and its role in biological activity in general and CNS activity in particular ever since Overton [58, 59] and Meyer [60, 61] discovered more than a century ago that narcotic potency in a set of congeners tends to increase as the oil/water partition coefficient P increases. Following its first use by Collander [62] and especially during the past 30 years, the log octanol/water partition coefficient, logPo/w, became a convenient standard measure for lipophilicity and one of the most informative physicochemical parameters used in the pharmaceutical and medicinal chemical fields [63]. The suitability of the octanol/water system to characterize CNS effects was underscored by a study of Franks and Lieb [64], who looked at anesthetic potency as a function of solvent/water partition coefficients and found that octanol gives an excellent correlation for all compounds and all animals over an extremely wide range. LogPo/w alone seems to have a more limited performance in predicting brain/ blood concentration ratios, but in combination with other parameters can still reasonably predict brain-blood partitioning [65–70]. In agreement with the structural aspects of the BBB described here, we also found a good correlation over a wide range of about eight log units between logPo/w and in vivo log permeability data of rat brain capillaries (log pBBB) for compounds that are not subject to active transport (Fig. 20.3) [63, 71]. However, increasing lipophilicity with the intent to improve membrane permeability might not only make chemical handling difficult, but might also increase the rate of oxidative metabolism by cytochromes P450 (CYPs) and other enzymes [72, 73]. In most cases, increasing lipophilicity increases potency and membrane permeability, but decreases dissolution and metabolic stability [74]. Hence, to improve bioavailability, the effects of lipophilicity on membrane permeability and first-pass metabolism have to be balanced [72, 75]. Furthermore, increasing lipophilicity tends to increase the volume of distribution [76] and tends to affect all other pharmacokinetic parame-


Fig. 20.3 In vivo log permeability coefficient of rat brain capillaries (log pBBB) as a function of log octanol/water partition coefficient (logPo/w). Names are shown for a few well known compounds, values denoted with a star are for guinea pig, and values denoted in italics are for cases where the log distribution coefficient measured at physiological pH was used (logDo/w). Strong deviants below the line, shown as diamonds, are known substrates for P-gp.

ters [72, 75]. Nonspecific toxicity is also well known to increase with logPo/w [77]; and King and Moffat have shown such a tendency even for approved hypnotics in humans [78]. It is difficult to formulate rules of general validity, but Hansch and co-workers [79] suggested that, when designing prospective new CNS drugs, the likelihood of success is better if one starts somewhere around an (apparent) calculated logPo/w = 2, and if CNS-related side-effects are to be avoided, it is probably better to avoid the 1 < logPo/w < 3 region. This has been referred to as the “rule of two” [80, 81]. During the past two decades, a variety of

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in silico models [82] and in vitro permeability assays [83] have been developed in attempts to characterize and predict BBB permeability and integrate such prediction into the early phases of drug development, together with various other considerations [57, 72, 77, 84]. Obviously, brain exposure can be increased not only by enhancing influx, but also by restricting efflux through the BBB [85]. For example, coadministration of a P-gp inhibitor can increase not only oral absorption, but also BBB permeability for P-gp substrates [86, 87]. Coadministration of the P-gp blocker valspodar has been shown recently not only to increase the brain levels of paclitaxel, but also to considerably improve its therapeutic effect on tumor volume in mice [88]. At present, the investigation of such possibilities are in the early exploratory phase only, but they might become useful in the future, maybe in combination with other, influx-enhancing approaches if toxicity in nontarget tissues and other problems can be avoided.

20.3.2 Quantifying Brain-Targeting: Site-Targeting Index and Targeting Enhancement Factors

Being able to quantitatively assess the site-targeting effectiveness of drug delivery systems or drug targeting in general is very important, and is particularly so for CNS-targeting. Recently, we proposed the use of targeting enhancement factors in the pharmacokinetic assessment of drug delivery systems [56, 89]. A sitetargeting index (STI) was defined as the ratio between the area under the concentration-time curve (AUC) for the drug itself at the targeted site and that at a systemic site, e.g., blood or plasma: STI AUCtarget =AUCblood

20:1

This index gives an accurate measure of how effectively the active therapeutic agent is actually delivered to its intended site of action. For example, as data in Table 20.1 indicate, some drugs (e.g., AZT, benzylpenicillin) are very ineffective in penetrating the BBB and reaching brain tissues: the AUCs of their brain concentrations are less than 1% of the corresponding blood values. For a drug delivery system, a site-exposure enhancement factor (SEF) was introduced to assess its effectiveness compared to the original drug itself. SEF measures the change in AUC of the active drug at the target site after administration of the delivery system, compared to administration of the drug alone: Delivery System

SEF AUCtarget

Drug Alone

=AUCtarget

20:2

Obviously, administration of equivalent (molar) doses is assumed for this definition. For slightly different doses, linearity between dose and AUC is a reasonable assumption; and the ratio of dose-normalized AUCs (AUC/dose) can be

20.3 Brain-Targeted Drug Delivery Table 20.1 Site-targeting indices (STI), site-exposure factor (SEF) and targeting enhancement factor (TEF) of the brain-targeting CDSs with available in vivo data. AUC = area under the concentration-time curve; S = spacer; AZT = azidovudine (3'-azido-3'-deoxythymidine); AZdU = 3'-azido-2',3'-dideoxyuridine; BP = benzylpenicillin (penicillin G). Compound Species and reference

D only Dose Time AUC (lmol (h) (lg h kg–1) ml–1) Brain

AZdU Mouse a) 197 [167] AZT 187 Mouse a) [167] Rat [159] 130 Rabbit 64 [156] BP, S = Me Rat [141] 60 Dog b) [142] 30 BP, S = Et Rat [141] 60 Dog b) [142] 30 Dexamethasone Rat [115] 20 Ganciclovir Rat c) [171] 80 a) b) c)

CDS STI (Eq. 20.1) Blood

AUC (lg h ml–1) Brain

STI

Blood

SEF

TEF

(Eq. 20.2)

(Eq. 20.3)

12

2.09

25.83

0.081

11.43

25.79

0.443

5.47

5.48

12

1.21

26.64

0.045

11.28

25.38

0.444

9.32

9.79

6 3

0.23 11.22

26.33 37.92

0.009 0.296

7.09 26.98

9.67 34.07

0.733 0.792

31.51 2.40

85.84 2.68

1 4

0.20 0.16

6.05 24.52

0.033 0.007

0.74 5.07

4.56 11.75

0.162 0.431

3.70 31.69

4.91 66.13

1 4

0.20 0.16

6.05 24.52

0.033 0.007

2.12 0.48

2.95 0.61

0.719 0.787

10.60 3.00

21.74 120.59

6

1.36

19.48

0.070

1.69

8.97

0.188

1.24

2.70

6

0.66

10.46

0.063

3.61

1.42

2.542

5.44

40.05

Brain and serum concentrations monitored. Cerebrospinal fluid and blood concentrations monitored. Brain and plasma concentrations monitored.

used in the definition of Eq. (20.2). Because a good delivery system not only increases exposure to the active agent at the target site, but also decreases the corresponding systemic exposure, a targeting enhancement factor (TEF) was introduced to quantify the relative improvement in the STI produced by administration of the delivery system, compared to administration of the drug itself: TEF STIDelivery System =STIDrug Alone

20:3

The TEF defined by Eq. (20.3) is the most rigorous measure that can be used to quantify the improvement in (pharmacokinetic) targeting produced by a delivery system. It compares not just concentrations, but concentrations along a time period, and it compares actual, active drug concentrations both at target and

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systemic sites. If: (1) the targeting or delivery (“carrier”) moiety produces no toxicity of its own, (2) the therapeutic effect is linearly related to AUCtarget, and (3) side-effects are linearly related to AUCblood (assumptions that are to a good extent satisfied under most conditions), then TEF also represents the enhancement produced in the therapeutic index (TI), which is usually defined as the ratio between the median toxic dose (TD50) and the median effective dose (ED50), TI = TD50/ED50: TEF TIDelivery System =TIDrug Alone

20:4

TEF, as defined here, represents the same ratio as a drug-targeting index used in a theoretical pharmacokinetic treatment [90]. A number of quantitative comparisons using these indices are presented in Table 20.1 for many of the CDSs discussed here.

20.4 Chemical Delivery Systems

CDS approaches provide novel, systematic methodologies for targeting active biological molecules to specific target sites or organs based on predictable enzymatic activation [71, 89, 91]. CDSs are inactive chemical derivatives of a drug obtained by one or more chemical modifications. They provide site-specific or site-enhanced delivery through sequential, multistep enzymatic and/or chemical transformations. The newly attached moieties are monomolecular units that are in general smaller or of similar size as the original molecule. Hence, the chemical delivery system term is used here in a stricter sense: they do not include systems in which the drug is covalently attached to large “carrier” molecules. In a general formalism, the bioremovable moieties introduced can be classified into two categories. Targetor (T) moieties are responsible for targeting, sitespecificity, and lock-in. Modifier functions (F1 . . . Fn) serve as lipophilizers, protect certain functions, or fine-tune the necessary molecular properties to prevent premature, unwanted metabolic conversions. CDSs are designed to undergo sequential metabolic conversions, first disengaging the modifier functions and then the targetor moiety, after it fulfills its site- or organ-targeting role. These transformations provide targeting (differential distribution) of the drug by either exploiting site-specific transport properties, such as those provided by the presence of the BBB, or by recognizing specific enzymes found primarily, exclusively, or at higher activity at the site of action. Three major CDS classes have been identified: (1) brain-targeting (enzymatic physical/chemical-based) CDSs, (2) site-specific enzyme-activated CDSs, and (3) receptor-based transient anchortype CDSs. By relying on these general concepts, successful deliveries have been achieved to the brain, eye, and lung, as summarized recently [89]. However, brain-targeting CDSs represent the most developed class, and here, we will review only aspects related to their development.

20.5 Brain-Targeting CDSs

20.5 Brain-Targeting CDSs 20.5.1 Design Principles

Brain-targeting chemical delivery systems represent the most developed CDS class. To obtain such a CDS, the drug is chemically modified to introduce the protective function(s) and the targetor (T) moiety. Upon administration, the resulting CDS is distributed throughout the body. Predictable enzymatic reactions convert the original CDS by removing some of the protective functions and modifying the T moiety, leading to a precursor form (T+-D), which is still inactive, but has significantly different physicochemical properties (Fig. 20.4). Whereas these intermediates are continuously eliminated from the “rest of the body”, at the targeted site, which has to be delimited by some specific membrane or other distribution barrier, efflux-influx processes are altered and provide a specific concentration. Consequently, release of the active drug essentially occurs only at the site of action. In other words, such a system exploits the idea that if a lipophilic compound enters the brain and is converted there into a hydrophilic molecule, it will be-

Fig. 20.4 Schematic representation of the multi-step sequential mechanism of brain-targeting CDSs designed to provide sustained and brain-specific activity.

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come “locked-in”: it will no longer be able to come out (Fig. 20.4). Targeting is assisted because the same conversion, as it takes place in the rest of the body, accelerates peripheral elimination, and further contributes to brain-targeting. In principle, many targetor moieties are possible for a general system of this kind [8, 92–96], but the one based on the 1,4-dihydrotrigonelline « trigonelline (coffearine) system, in which the lipophilic 1,4-dihydro form (T) is converted in vivo to the hydrophilic quaternary form (T+; Fig. 20.5), proved the most useful. This conversion takes place easily everywhere in the body, because it is closely related to the ubiquitous NADH « NAD+ coenzyme system associated with numerous oxidoreductases and cellular respiration [97, 98]. Because oxidation takes place with direct hydride transfer [99] and without generating highly active or reactive radical intermediates, it provides a nontoxic targetor system [100]. Furthermore, it was shown [101] that the trigonellinate ion formed after cleavage of the CDS undergoes rapid elimination from the brain, most likely by involvement of an active transport mechanism that eliminates small organic ions, and therefore, the T+ moiety formed during the final release of the active drug D from the charged T+-D form will not accumulate within the brain [101, 102]. Whereas the charged T+-D form is locked behind the BBB into the brain, it is easily eliminated from the body as a result of the acquired positive charge, which enhances water solubility. After a relatively short time, the delivered drug D (as the inactive, locked-in T+-D) is present essentially only in the brain, providing sustained and brain-specific release of the active drug. It has to be emphasized again that the system not only achieves delivery to the brain, but it

Fig. 20.5 The 1,4-dihydrotrigonellinate « trigonellinate redox system used in brain-targeting CDSs, in analogy to the ubiquitous NADH « NAD+ coenzyme system.


achieves preferential delivery, which means brain-targeting. This should allow smaller doses and reduce peripheral side-effects. Furthermore, because the “lock-in” mechanism works against the concentration gradient, the system also provides more prolonged effects. Consequently, these CDSs can be used not only to deliver compounds that otherwise have no access to the brain, but also to retain lipophilic compounds within the brain, as has indeed been achieved, for example, with a variety of steroid hormones. During the past two decades, the CDS approach has been explored with a wide variety of drug classes, e.g., biogenic amines: phenylethylamine [103–106], tryptamine [107, 108], steroids: testosterone [96, 109–111], progestins [112], progesterone [113], ethinyl estradiol [114], dexamethasone [115, 116], hydrocortisone [96], alfaxalone [117], estradiol [118–138], anti-infective agents: penicillins [139– 142], sulfonamides [143], antivirals: acyclovir [91, 144], trifluorothymidine [145, 146], ribavirin [147–149], Ara-A [147], deoxyuridines [150–152], 2'-F-5-methylarabinosyluracil [153], antiretrovirals: zidovudine (AZT) [154–168], 2',3'-dideoxythymidine [102], 2',3'-dideoxycytidine [169, 170], ganciclovir [171], cytosinyl-oxathiolane [172], anticancer agents: lomustine (CCNU) [173, 174], HECNU [174], chlorambucil [175], neurotransmitters: dopamine [176–180], GABA [181, 182], nerve growth factor (NGF) inducers: catechol derivatives [183, 184], anticonvulsants: GABA [182], phenytoin [185], valproate [186], stiripentol [187], the calcium channel antagonist felodipine [188], MAO inhibitors: tranylcypromine [189, 190], anti-dementia drugs (cholinesterase inhibitors): tacrine [191, 192], antioxidants: LY231617 [193], chelating agents: ligands for technetium complexes [194], nonsteroidal anti-inflammatory drugs (NSAIDs): indomethacin [195], naproxen [195], anesthetics: propofol [196], nucleosides: adenosine [197], and peptides: tryptophan [198, 199], Leu-enkephalin analogs [200, 201], thyreotropin-releasing hormone (TRH) analogs [202–208], kyotorphin analogs [209, 210], and S-adenosyl-l-methionine (SAM) [211]. Selected examples will be briefly summarized together with representative physicochemical properties, metabolic pathways, and pharmacological data.

20.5.2 Zidovudine-CDS

Infection by human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS, has many devastating consequences, including encephalopathy with subsequent dementia. Many AIDS patients have demonstrable neurological damage related to this syndrome. Antiviral agents need to reach the CNS in therapeutically relevant concentrations to adequately treat AIDS dementia, but most potentially useful drugs cannot penetrate through the BBB. Zidovudine (3'-azido-3'-deoxythymidine, azidothymidine; AZT) was the first drug approved for the treatment of AIDS. This modified riboside has been shown to be useful in improving the neuropsychiatric course of AIDS encephalopathy in a few patients, but the doses required for this improvement usually precipitated severe anemia.

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Following the increasing occurrence of AIDS, AZT-CDS was investigated in a number of laboratories to enhance the access of AZT to brain tissues [154–168]. AZT-CDS is relatively easily accessible synthetically; and it is a crystalline solid which is stable at room temperature for several months when protected from light and moisture. It is relatively stable in pH 7.4 phosphate buffer, but rapidly oxidizes in enzymatic media. In addition, T+-AZT, the depot form of AZT, was shown to gradually release the parent compound. The ability of AZT-CDS to provide increased brain AZT levels has been demonstrated in a number of different in vivo animal models. The corresponding AUC data for mice [167], rats [159], and rabbits [156] are presented in Table 20.1. In all species, AZT-CDS provided substantially increased brain-targeting of AZT. For example, the TEF was 86 in rats. It is interesting to note that the relatively large difference in the TEF values for different species is mainly due to the variability of the brain-STI of AZT itself, AZT-CDS gave very similar values in all three species. In rabbits, the brain/blood ratio never exceeded 1 after AZT administration, indicating that the drug is always in higher concentration in the blood than in the brain, but it approached 3 at 1 h after AZT-CDS administration. In dogs, AZT levels were 46% lower in blood, but 175–330% higher in brain after AZTCDS administration than after AZT administration [160]. Furthermore, in vitro experiments found AZT-CDS not only more effective in inhibiting HIV replication than AZT itself, presumably due to hydrolysis, but also less toxic to host lymphocytes [159, 162, 163]. In conclusion, a number of different studies found an improved delivery to the brain and a decreased potential for dose-limiting side-effects, suggesting that AZT-CDS may become useful in the treatment of AIDS encephalopathy and the related dementia.

20.5.3 Ganciclovir-CDS

A CDS approach also allowed enhanced brain-delivery for ganciclovir (Fig. 20.6), another antiviral agent that may be useful in treating human encephalitic cytomegalovirus (CMV) infection [171]. CMV infections are common and usually benign; however, human CMV occurs in 94% of all patients suffering with AIDS and it has been implicated as a deadly cofactor. Whereas positive results could be achieved in the treatment of the associated retinitis with ganciclovir, they were much harder to come by in the treatment of encephalitic cytomegalic disease. A brain-targeted CDS was therefore investigated; and improved organ selectivity was demonstrated. As shown in Fig. 20.6 and Table 20.1, a ca. 5-fold increase in the relative brain exposure to ganciclovir and a simultaneous 7-fold decrease in blood exposure was achieved with the CDS in rats.


Fig. 20.6 Ganciclovir (GV) concentrations in brain and plasma as a function of time after an i.v. dose of 80 lmol kg–1 of GV or GV-CDS in rats. Data are means ± SEM.

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20.5.4 Benzylpenicillin-CDS

The treatment of various bacterial infections localized in brain and cerebrospinal fluid is often hindered by the poor CNS penetration of b-lactam antibiotics, including penicillins and cephalosporins. In addition, because these antibiotics return back to the blood from the CNS (active transport may be involved), simple prodrug approaches could not provide real solutions. Consequently, a number of CDSs have been synthesized and investigated [139–142]. Because in these structures the targetor moiety had to be coupled to an acid moiety, a spacer function had to be inserted between the targetor and the drug. This renders additional flexibility to the approach, and several possibilities were investigated. Benzylpenicillin (BP)-CDS was one of the first cases where the influence of such functions on the stability and delivery properties of CDSs could be investigated. Systematic studies of the effects of various modifications have been performed since then [93, 201]. In rats, i.v. administration of BP-CDS with an ethylene 1,2-diol spacer (S = Et) gave an AUC-based brain-exposure enhancement factor for BP of around 11 (see Table 20.1) [141]. From the several coupling possibilities investigated, diesters of methylene diol and ethylene 1,2-diol proved worthy of further investigation, but ester-amide combinations did not prove successful. Consequently, the diesters have also been investigated in rabbits and dogs [142]. Both in rabbits and in dogs, i.v. administration of equimolar doses of CDSs provided BP levels in brain and cerebrospinal fluid (CSF) that were substantially higher and more prolonged than in the case of parent drug administration. In this study, the diester of methylene diol provided more significant increase in BP brain exposure (SEF = 32); and the diester of ethylene 1,2-diol gave better targeting, but released lower drug concentrations due to the formation of an inactive hydroxyethyl ester. Because dogs were not sacrificed, only blood and CSF data were collected (Table 20.1); and these indicated better CSF penetration in dogs than in rabbits. Although parenteral injection of BPs may sometimes cause undesired side-effects, such as seizures, the high CNS levels obtained in dogs and rabbits after CDS administration were not accompanied by any toxic side-effects [142]. Similarly, although the delivered compound exhibited toxicity in the rotorod test at 0.5 h following a 300 mg kg–1 i.p. dose, felodipine-CDS displayed no toxicity at either 0.5 h or 4.0 h after a 100 mg kg–1 i.p. dose or 0.5 h for a 300 mg kg–1 i.p. dose [188]. Some toxicity, which is not surprising considering the toxicity of the delivered compound itself, was observed at 4.0 h for the 300 mg kg–1 CDS dose.


20.5.5 Estradiol-CDS

Estradiol-CDS (E2-CDS; Estredox) is in the most advanced investigation stage of all CDSs (phase I/II clinical trials). Estrogens are endogenous hormones that produce numerous physiological actions and are among the most commonly prescribed drugs in the United States, mainly for hormone replacement therapy (HRT) in postmenopausal women and as a component of oral contraceptives [212]. They are lipophilic steroids that are not impeded in their entry to the CNS, and hence, they can readily penetrate the BBB and achieve high central levels after peripheral administration. However, estrogens are poorly retained within the brain. Therefore, frequent or sustained (e.g., transdermal) doses have to be administered to maintain therapeutically significant concentrations. Menopause-related estrogen depletion is more than likely associated with a variety of symptoms that range the gamut from vasomotor complaints to cognitive deficits. Traditional HRT may alleviate many of these complications, but because of increased risks for cancer and other metabolic diseases, many women avoid treatment. Constant peripheral exposure to estrogens has been related, however, to a number of pathological conditions including cancer, hypertension, stroke, and altered metabolism [213–217]. The Heart and Estrogen/Progestin Replacement Study (HERS) has already suggested that, contrary to earlier expectations, HRT does not benefit the incidence of coronary heart disease (CHD) [218, 219]. As a very important development, two large-scale parallel randomized, doubleblind, placebo-controlled clinical trials were undertaken within the Women’s Health Initiative (WHI) to determine whether conjugated equine estrogen (CEE) alone (in women with prior hysterectomy) or in combination with progestin (medroxy-progesterone acetate) reduces cardiovascular events in postmenopausal women; and both were halted early because of unfavorable outcomes. The estrogen plus progestin component, which had a planned duration of 8.2 years and recruited 16 608 postmenopausal women, was stopped prematurely because it indicated an unacceptably increased risk for invasive breast cancer (26%) [220, 221]. It also indicated a 41% increase for stroke and a 29% increase for CHD. The estrogen alone component, which enrolled 10 739 postmenopausal women, was also ended early as the use of CEE was found to increase the risk of stroke and not affect the incidence of CHD (but decrease the risk of hip fracture) in postmenopausal women with prior hysterectomy [222]. Considering all this and that the approx. 30% increase in the risk of breast cancer caused by taking estrogen is suspected to rise to 50% if taken for more than 10 years [223], there is little justification now for systemic HRT. This is an important decision, since in industrialized nations, the average woman spends around a third of her life in the postmenopausal stage (menopause occurs at an average age of 51) [224]. As the CNS is the target site for many estrogenic actions [225–227], brain-targeted delivery may provide safer and more effective agents in many cases. With the recent unraveling of the many roles estrogen plays in males [228], there might be at least as many therapeutic possibilities in males as in females.

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Estrogen CDSs could be useful in reducing the secretion of luteinizing hormone-releasing hormone (LHRH), and hence, in reducing the secretion of LH and gonadal steroids. As such, they could be employed to achieve contraception and to reduce the growth of peripheral steroid-dependent tumors, such as those of the breast, uterus, and prostate, and to treat endometriosis. They also could be useful in stimulating male and female sexual behavior, and in the treatment of menopausal vasomotor symptoms (“hot flushes”) [229]. Other potential uses are in neuroprotection, in the reduction of body weight, and in the treatment of depression, schizophrenia [226], and various types of dementia, including Alzheimer’s disease [215, 230–237]. Estradiol (E2) is the most potent natural steroid. Because it contains two hydroxy functions (one in the phenolic 3 position and one in the 17 position), three possible CDSs can be designed by attaching the targetor at 17, at 3, or at both positions. Attachment at either position, but especially at the 17 position, should greatly decrease the pharmacological activity of E2, because these esters are known not to interact with estrogen receptors [238]. Since its first synthesis in 1986 [118], E2-CDS has been investigated in several models [119–129, 131–138]. In vitro studies with rat organ homogenates as the test matrix indicated a half-life of 156.6 min, 29.9 min, and 29.2 min for T at the 17 position, in plasma, liver, and brain homogenates, respectively [118]. Thus, E2-CDS is converted to the corresponding quaternary form (T+-E2) faster in tissue homogenates than in plasma. This is consistent with the hypothesis of a membrane-bound enzyme, such as the members of the NADH transhydrogenase family, acting as oxidative catalyst. These studies also indicated a very slow production of E2 from T+-E2, suggesting a possible slow and sustained release of estradiol from brain deposits of T+-E2. A rat study that used a precolumn-enriching HPLC system for increased analytical accuracy [125] found that E2 released from the T+-E2 intermediate formed after i.v. E2-CDS administration has an elimination half-life of more than 200 h, and brain E2-levels are elevated 4- to 5-fold longer after administration than after simple estradiol treatment [125]. Proving effective targeting, another study also found that steroid levels between 1 day and 16 days after E2-CDS treatment were more than 12-fold higher in brain samples than in plasma samples [129]. Studies in orchidectomized rats proved that a single i.v. injection of E2-CDS (3 mg kg–1) suppressed LH secretion by 88%, 86%, and 66% relative to DMSO controls at 12, 18, and 24 days, respectively, and that E2 levels were not elevated relative to the DMSO control at any sampling time [121]. A single i.v. administration of doses as low as 0.5 mg kg–1 to ovariectomized rats induced prolonged (3–6 weeks) pharmacological effects as measured by LH suppression [119, 121, 129], reduced rate of weight gain [122, 127–129], or, in castrated male rats, reestablishment of copulatory behavior [120]. E2-CDS also provided very encouraging neuroprotective effects. In ovariectomized rats, pretreatment with E2-CDS decreased the mortality caused by middle cerebral artery occlusion (MCAO) from 65% to 16% [135]. E2-CDS reduced the area of ischemia by 45–90% or 31%, even if administered after MCAO by 40 min or 90 min, respectively. Evi-


dence was also found that treatment with E2-CDS can protect cholinergic neurons in the medial septum from lesion-induced degeneration [136]. A recent study not only confirmed that E2-CDS can radically reestablish copulatory behavior in castrated male rats, but also found a dramatic effect on the sexual behavior of female rats (Fig. 20.7) [138]. Considering the complexity and therapeutic potential of female sexual dysfunction [239, 240], this is a surprising, but significant discovery. Because female sexual dysfunction is more complex than male sexual dysfunction, such a strong response caused by administration of an estradiol derivative compound alone is quite unexpected. There is no consensus in the corresponding literature, but there is evidence that the decline of female sexual functioning with the natural menopausal transition is more closely related to decreasing estradiol (E2) levels than to androgen levels [241]. In the United States, more women (43%, about 40 million) than men (31%) experience some form of sexual disorder, and in contrast to men, the distribution of female sexual dysfunction is fairly even among women ranging over 18–59 years of age. E2-CDS reestablished the copulatory behavior from day 7 to day 28 in orchidectomized males after single i.v. doses of 0.3 mg kg–1 and 3.0 mg kg–1 [138]. LH suppression started on day 1 and lasted until day 7 and day 28, respectively. In ovariectomized female rats, sexual activity was monitored through the lordosis behavior. Animals were treated daily i.v. for 5 days with E2-CDS, estradiol 17-benzoate (E2-Bz), or vehicle, respectively. At a dose of only 0.01 mg kg–1, E2-CDS already normalized sexual behavior (Fig. 20.7). At the 0.03 mg kg–1 dose level, the effect of E2-Bz was 10 days shorter than that of E2-CDS. LH suppression lasted for up to 18 days at the 0.03 mg kg–1 dose and for up to 10 days at the 0.01 mg kg–1 and 0.003 mg kg–1 doses. However, there was no significant decrease in LH levels after E2-Bz treatment. The low plasma estradiol levels indicated a fast rate of peripheral elimination in both males and females. These results confirm again the potential of E2-CDS to cause significantly higher and more prolonged effects than the isolipophilic E2-Bz. Other encouraging results obtained in various animal models and earlier phase I/II clinical trials have been reviewed [91, 126, 131, 132, 134]. More recently, a new, more convenient buccal formulation tablet, which should be dissolved in the cheek area of the mouth and not swallowed, has been developed and evaluated in two phase I/II clinical studies. One was a single-center, openlabel, pilot phase II study for postmenopausal symptoms using circulating LH and FSH as biomarker in 12 healthy postmenopausal female volunteers not on ERT. Following a single E2-CDS administration (2.5 mg or 5.0 mg), sustained plasma estradiol levels were maintained for up to 3 days, confirming the release of active E2 from the intermediate “locked-in” the brain; and both LH and FSH blood levels were suppressed in a statistically significant manner (paired t-test analysis), as indicated by concentration and AUC24 h data [242]. The other study was a single-center, open-label, parallel-group phase Ib/II study in 12 healthy postmenopausal female subjects not on ERT/HRT. Twelve subjects in two groups of six received one buccal tablet containing 2.86 mg E2-CDS once daily

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Fig. 20.7 Effect of different doses of E2-CDS and the isolipophilic E2-benzoate on the sexual behavior of ovariectomized female rats, as measured by the lordosis quotient. Lordosis is the vertebral dorsiflexion performed by female quadrupeds in response to adequate stimuli from a reproductively competent male; and the lordosis quotient is calculated as the number of lordosis per ten mounts (%). E2-CDS was administered for 5 days i.v. dissolved in 27% HPbCD solution. Data are means ± SE for 8–12 animals per group (* P < 0.05, ** P < 0.01, *** P < 0.001; using Mann-Whitney U test).


Fig. 20.8 Baseline-adjusted total E2 concentrations for the whole study period following administration of 2.86 mg E2-CDS as a buccal tablet once daily (group A) or once every other day (group B). Data are means ± SD for six subjects in group B and five subjects in group A, where the data for one subject, which had about 10-fold higher E2 levels than the other subjects, are not included in this graphic.

for 10 days (group A) or once every second day for 13 days (group B). Buccal E2CDS administered daily or every other day was well-tolerated. Serum E2 levels were higher for the daily regimen (group A), and differences were significant (Fig. 20.8) [243]. Both dosing regimens caused significant LH- and FSH-suppression, providing evidence of CNS penetration. In conclusion, clinical evaluations suggested a potent central effect coupled with only marginal tendencies to elevate systemic estrogen levels. Furthermore, peripheral levels seem to be susceptible to easy manipulation through dosing amount and frequency, and therefore, this approach could represent the ideal estrogen replacement therapy. The data suggest that E2-CDS may be a useful and safe therapy for menopausal symptoms, estrogen-dependent cognitive deficits, including Alzheimer’s disease, male and female sexual dysfunction, and possibly even neuroprotection.

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20.5.6 Cyclodextrin Complexes

Unfortunately, the same physicochemical characteristics that allow successful chemical delivery also complicate the development of acceptable pharmaceutical formulations. Increased lipophilicity allows partition into deep brain compartments, but also confers poor aqueous solubility. The oxidative lability, which is needed for the lock-in mechanism, and the hydrolytic instability, which releases the modifier functions or the active drug, combine to limit the shelf-life of the CDS. Cyclodextrins may provide a possible solution. They are torus-shaped oligosaccharides that contain various numbers of a-1,4-linked glucose units (6, 7, 8 for a-, b-, c-cyclodextrin, respectively). The number of units determines the size of a cone-like cavity into which various compounds can enter and form stable complexes [244–247]. Formation of the host-guest inclusion complex generally involves only the simple spatial entrapment of the guest molecule without the formation of covalent bonds [248]. Complexation with 2-hydroxypropyl-b-cyclodextrin (HPbCD) provided important advances in the formulation of E2-CDS [249]. HPbCD was selected based on its low toxicity observed using various administration routes and based on the fact that alkylation or hydroxyalkylation of the glucose oligomer can disrupt hydrogen bonding and provide increased water solubility for the compound and its inclusion complexes [250–253]. Indeed, the aqueous solubility of E2-CDS was enhanced about 250 000-fold in a 40% (w/v) HPbCD solution (from 65.8 ng ml–1 to 16.36 mg ml–1). The phase solubility diagram indicated that a 1 : 1 complex forms at low HPbCD concentration, but a 1 : 2 complex occurs at higher HPbCD concentration. The stability of E2-CDS was also significantly increased, allowing formulation in acceptable form. The rate of ferricyanide-mediated oxidation, a good indicator of oxidative stability, was decreased about 10-fold and shelf life was increased about 4-fold, as indicated by t90 and t50 values in a temperature range of 23–80 8C [249]. The cyclodextrin complex even provided better distribution by preventing retention of the solid material precipitated in the lung. Promising results were also obtained for testosterone-CDS [111], lomustine-CDS [174], and BP-CDS [254]. For the latter, aqueous solubility was enhanced about 70 000-fold in a 20% (w/v) HPbCD solution (from 50–70 ng ml–1 to 4.2 mg ml–1), and stability was also increased.

20.6 Molecular Packaging

The CDS approach also has been extended to achieve successful brain deliveries of a Leu-enkephalin analogs [200, 201], thyreotropin-releasing hormone (TRH) analogs [202–208], and kyotorphin analogs [209, 210]. Neuropeptides, peptides that act on the brain or spinal cord, represent the largest class of transmitter substance and have considerable therapeutic potential [209, 255, 256]. The number of identified endogenous peptides is growing exponentially, and recently, al-


most 600 sequences for neuropeptides and related peptides have been listed [257]. However, the delivery of peptides through the BBB is an even more complex problem than the delivery of other drugs, because they can be rapidly inactivated by ubiquitous peptidases [31, 32, 258–260]. Therefore, for a successful delivery, three issues have to be solved simultaneously: (1) enhance passive transport by increasing the lipophilicity, (2) ensure enzymatic stability to prevent premature degradation, and (3) exploit the lock-in mechanism to provide targeting. The solution obtained as a generalization of the CDS approach is a complex molecular packaging strategy, in which the peptide unit is part of a bulky molecule dominated by lipophilic-modifying groups that direct BBB penetration and prevent recognition by peptidases [261]. Such a brain-targeted packaged peptide delivery system contains the following major components: the redox targetor (T), a spacer function (S), consisting of strategically used amino acids to ensure timely removal of the charged targetor from the peptide, the peptide itself (P), and a bulky lipophilic moiety (L) attached through an ester bond or sometimes through a C-terminal adjuster (A) at the carboxyl terminal to enhance lipid solubility and to disguise the peptide nature of the molecule. Obviously, to achieve delivery and sustained activity with such complex systems, it is very important that the designated enzymatic reactions take place in a specific sequence. On delivery, the first step must be the conversion of the targetor to allow for lock-in. This must be followed by removal of the L function to form a direct precursor of the peptide that is still attached to the charged targetor. Subsequent cleavage of the targetor-spacer moiety finally releases the active peptide.

20.6.1 Leu-Enkephalin Analogs

Since the discovery of endogenous peptides with opiate activity [262], many potential roles have been identified for these substances [263, 264]. Possible therapeutic applications could extend from the treatment of opiate dependence to numerous other CNS-mediated dysfunctions. Opioid peptides are implicated in regulating neuroendocrine activity [265–267], motor behavior [268], seizure threshold [269, 270], feeding and drinking [271], mental disorders [263, 272], cognitive functions [263, 273], cardiovascular functions [274, 275], gastrointestinal functions [263, 276], and sexual behavior and development [264]. However, analgesia remains their best known role; and it is commonly used to evaluate endogenous opioid peptide activity. Whereas native opioid peptides could alter pain threshold after intracerebroventricular (i.c.v.) dosing, they were ineffective after systemic injection. It was reasonable, therefore, to attempt a brain-targeted CDS approach. The first successful delivery and sustained release was achieved for Tyr-D-AlaGly-Phe-D-Leu (DADLE), an analog of Leu-enkephalin, a naturally occurring lin-

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ear pentapeptide (Tyr-Gly-Gly-Phe-Leu) that binds to opioid receptors [200]. In rat brain tissue samples collected 15 min after i.v. CDS administration, electrospray ionization mass spectrometry clearly showed the presence of the locked-in T+-D form at an estimated concentration of 600 pmol g–1 of tissue. The same ion was absent from the sample collected from the control animals treated with the vehicle solution only. To optimize this delivery strategy, an effective synthetic route was established for peptide CDSs and the role of the spacer and the lipophilic functions was investigated [201]. Four different CDSs were synthesized by a segment-coupling method. Their i.v. injection produced a significant and long-lasting (> 5 h) response in rats monitored by classic [277] tail-flick latency test. Compared with the delivered peptide itself, the packaged peptide and the intermediates formed during the sequential metabolism had weak affinity to opioid receptors. The antinociceptive effect was naloxone-reversible and methylnaloxonium-irreversible. Because quaternary derivatives such as methylnaloxonium are unable to cross the BBB, these techniques [201, 278] are used to prove that central opiate receptors are responsible for mediating the induced analgesia. It could be concluded, therefore, that the peptide CDS successfully delivered, retained, and released the peptide in the brain. The efficacy of the CDS package was strongly influenced by modifications of the spacer (S) and lipophilic (L) components, proving that they have important roles in determining molecular properties and the timing of the metabolic sequence. The bulkier cholesteryl group used as L showed a better efficacy than the smaller 1-adamantaneethyl, but the most important factor for manipulating the rate of peptide release and the pharmacological activity turned out to be the spacer (S) function: proline as spacer produced more potent analgesia than alanine.

20.6.2 TRH Analogs

A similar strategy was used [202–206] for the CNS delivery of a thyreotropin-releasing hormone (TRH; Glp-His-Pro) analog (Glp-Leu-Pro) in which histidine (His2) was replaced with leucine (Leu2) to dissociate CNS effects from thyreotropin-releasing activity [279]. Such compounds can increase extracellular acetylcholine (ACh) levels, accelerate ACh turnover, improve memory and learning, and reverse the reduction in high affinity choline uptake induced by lesions of the medial septal cholineric neurons [280, 281]. Therefore, as reviewed in the literature [282, 283], these peptides are potential agents for treating motor neuron diseases [284], spinal cord trauma [285], or neurodegenerative disorders such as Alzheimer’s disease [286], and may also have a potential cytoprotective role [287]. Because the peptide that has to be delivered has no free -NH2 and -COOH termini, a precursor sequence (Gln-Leu-Pro-Gly) that will ultimately produce the final TRH analog was packaged for delivery. Therefore, two additional steps had to be included in the metabolic sequence: one where the C-terminal adjuster glycine is cleaved by peptidyl glycine a-amidating monooxygenase (PAM) to


form the ending prolinamide, and one where the N-terminal pyroglutamyl is formed from glutamine by glutaminyl cyclase [202]. In summary, the following sequential biotransformation has to take place after delivery to the brain: first, lock-in by oxidation of the dihydrotrigonellyl (T) to the corresponding pyridinium salt, then removal of cholesterol, oxidation of glycine by peptidyl glycine aamidating monoxygenase to prolineamide, cleavage of the targetor-spacer combination, and finally, cyclization of glutamine by glutaminyl cyclase to pyroglutamate. Selection of a suitable spacer proved also important for the efficacy of TRHCDSs as measured by the decrease in the barbiturate-induced sleeping time in mice, an interesting and well documented effect of this neuropeptide [288, 289]. After i.v. administration of 15 lmol kg–1, the Leu2-TRH analog itself produced only a slight decrease of 17 ± 7%, compared to the vehicle control. Equimolar doses of the CDS compounds with Pro, Pro-Pro, and Pro-Ala spacers produced statistically significant (P < 0.05) decreases of 47 ± 6%, 51 ± 7%, and 56 ± 4%, respectively [204]. A Nva2-TRH analog, in which norvaline replaced leucine was also used in such an approach with a Pro-Pro spacer, and very similar results were obtained with a dose of 20.7 lmol kg–1 in Swiss-Webster mice [206]. Treatment with a Leu2-TRH-CDS also significantly improved memory-related behavior in a passive avoidance paradigm in rats bearing bilateral fimbrial lesions without altering thyroid function [203]. Because the amide precursor was found to be susceptible to deamination by TRH-deaminase, a side-reaction process competitive with the designed-in cleavage of the spacer-Gln peptide bond, another analog of TRH was also examined [205]. The Pro-Gly C terminal was replaced by pipecolic acid (Pip) to obtain a TRH analog that is active in the carboxylate form and, hence, to eliminate the need for formation of the amide. The CDS was prepared by a 5+1 segment-coupling approach and the cholesteryl ester of pipecolic acid was prepared using either Fmoc (fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonyl) as protection. Pharmacological activity was assessed by antagonism of barbiturate-induced sleeping time in mice; and the pipecolic acid analog was found to be even somewhat more effective than the previous package.

20.6.3 Kyotorphin Analogs

Finally, a kyotorphin analog (Tyr-Lys; YK) that has activity similar to kyotorphin itself [290] was also successfully delivered [209, 210]. Kyotorphin (Tyr-Arg) is an endogenous neuropeptide that exhibits analgesic action through the release of endogenous enkephalin; and its analgesic activity is about four times larger than that of Met-enkephalin [291]. It also has other effects that may prove useful, such as seizure protective effects [292] or effects on nitric oxide synthase (NOS) [293–295]. Because this peptide contains a free amine residue, and because preliminary studies indicated that such ionizable functions might prevent

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Fig. 20.9 Reversal of the analgesia produced by 22.3 lmol kg–1 CDS(PP) and BTRA of the kyotorphin analog YK administered i.v. by naloxone (6.1 lmol kg–1, s.c.) administered 0.5 h later. Data are means ± SE of six rats for each group.

successful delivery, this free amine functionality was additionally “packaged” by attachment of a Boc group. It was assumed that this attachment is bioconvertible and the lock-in mechanism will allow time for its enzymatic removal. Because during the synthesis of this CDS package, a- and e-amine functions had to be differentially functionalized, combined liquid-phase Fmoc and Boc chemistry was employed. With this synthetic method at hand, a number of various spacers were explored for this delivery system (Pro, Pro-Ala, Pro-Pro); and the double proline CDS(PP) provided the best pharmacological effect amongst them (Fig. 20.9). The corresponding CDS showed good activity on the rat tailflick latency test, even with a single proline spacer. Activity was already significant at a 3 lmol kg–1 (1.0 mg kg–1) dose and leveled off at about 22 lmol kg–1. This represents an improvement of about two orders of magnitude compared to the ~ 200 mg kg–1 dose necessary to observe activity when the peptide itself is given intravenously. Several intermediates and building blocks were also studied, but only administration of the whole molecular package produced significant pharmaceutical response, confirming that only the designed metabolic sequence as a whole is effective in delivering peptides across the BBB.


20.6.4 Brain-Targeted Redox Analogs

Kyotorphin analogs also served for the design and evaluation of a novel and conceptually different method that can provide brain-targeted activity for peptides containing amino acids with basic side-chains (lysine, arginine) [210]. Within this approach, the targetor T moiety, which by conversion to its charged T+ form is responsible for the lock-in mechanism, is not attached to the peptide from outside, but it is integrated within novel redox amino acids building blocks that replace the original basic amino acid of the active peptide. Consequently, isosteric/isoelectronic analogy between the original and the redox side-chain and not cleavage of the redox targetor is expected to provide activity. A comparison of the structures of the classic brain-targeted CDS and the novel brain-targeted redox analog (BTRA) used for the present kyotorphin analog (YK) can be seen in Fig. 20.9. The e-amine function of lysine can be directly replaced by the nicotinamide function, using the Zincke procedure to form pyridinium salts [199, 210, 296–298]. As shown in Fig. 20.9, both the CDS and the BTRA produced significant and prolonged analgesic effect in rats; and both could be reversed by naloxone, demonstrating again that central opiate receptors must be responsible for mediating the induced analgesia. Such novel amino acids may provide both effective replacement and targeting for other lysine or arginine containing peptides. In a copycat design, a BTRA approach has also been attempted for TRH by replacing its His2 residue with a redox analog [207, 208] as this His is believed to be essential for endocrine, TSH-releasing activity, but not for CNS effects [299]. These analogs lost endocrine activity as measured by binding affinity to receptor from rat brain labeled by (3H)(3-Me-His2)TRH, but some of them seemed to maintain analeptic activity, as measured by the decrease in the barbiturate-induced sleeping time in mice. As the chain length separating the redox moiety from the amino acid backbone increased, the potency decreased. Hence, molecular packaging of peptides is a rational drug design approach that achieved the first documented non-invasive brain delivery of these important biomolecules in pharmacologically significant amounts. Because drug delivery is the weakest link of the peptide drug industry [300], this approach may represent an important step toward future generations of high-efficiency neuropharmaceuticals obtained from biologically active peptides.

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21 Drug Delivery to the Brain by Internalizing Receptors at the Blood-Brain Barrier Pieter J. Gaillard, Corine C. Visser, and Albertus (Bert) G. de Boer

21.1 Introduction

Drug delivery to the brain is still a major obstacle for the effective treatment of brain diseases. However, effective and safe ways of targeting drugs to the entire brain have been developed by applying delivery systems directed to endogenous receptor-mediated uptake mechanisms present at the cerebral capillaries. Such systems have been shown to be effective up to primates, but not yet in humans. In this chapter we focus on the well characterized transferrin system and the more recently described systems that use the low-density lipoprotein receptor-related protein 1 (LRP1) receptor, the LRP2 receptor (also known as megalin and glycoprotein 330) or the diphtheria toxin receptor (DTR, which is the membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor, HB-EGF). The possibilities and limitations of these systems together with their future human application are discussed. For many diseases of the brain, such as Alzheimer’s disease, Parkinson’s disease, stroke, depression, schizophrenia, epilepsy and migraine headache, the drugs on the market are far from being ideal, let alone curative, drugs. A significant part of the problem is the poor blood-brain barrier penetration of most of the drugs in development against neuronal targets for treatment of these disorders. This includes approximately 98% of the small molecules and nearly 100% of large molecules, such as recombinant proteins or gene-based medicines [1]. Therefore, much effort is put towards delivery and targeting of drugs to the brain. As a result, various drug delivery and targeting strategies are currently being developed. We discuss in this chapter the possibilities to deliver drugs to the brain via the vascular route by receptor-mediated uptake/transcytosis into the (human) brain.


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21 Drug Delivery to the Brain by Internalizing Receptors at the Blood-Brain Barrier

21.2 Blood-Brain Barrier Transport Opportunities

The development and the anatomy of the blood-brain barrier have been treated elsewhere in this book (see Chapters 1 to 3). Here we want to state that the blood-brain barrier, located in the brain capillaries, can be considered as an interface between blood and brain regulating the homeostasis of the brain and limiting the transport of many compounds into the brain (Fig. 21.1). Next to the blood-brain barrier, there is the blood-cerebrospinal fluid barrier (B-CSFB) that is located in the choroid plexus. Although choroid plexus epithelial cells have microvilli, it can be stated that, with respect to intravascular administration of drugs, the surface area of the blood-brain barrier is several decades larger than the B-CSFB. For an extensive review of the concept and the properties of the blood-brain barrier, we refer to preceeding chapters in this book (Chapters 4 to 9) and the literature [2–9]. In addition, important contributions have been made to the topic of drug delivery to the brain (see Chapters 18 to 20); [10–12]. Here we want to focus on receptor-mediated drug delivery to the brain. One of the most important properties of the brain capillary endothelial cells (BCEC) of the blood-brain barrier with respect to drug transport to the brain is that they have specific characteristics, such as tight junctions, which prevent paracellular transport of small and large (water-soluble) compounds from blood to the brain [9, 13, 14]. Furthermore, transcellular transport from blood to brain is limited as a result of low vesicular transport, high metabolic activity and a

Fig. 21.1 Schematic representation of the blood-brain barrier, the bloodcerebrospinal fluid (CSF) barrier and the brain-CSF barrier. The blood-brain barrier has the largest surface area and is, therefore, considered to be the most important influx barrier for solutes to reach the brain.

21.2 Blood-Brain Barrier Transport Opportunities

lack of fenestrae [5]. These specific characteristics of the blood-brain barrier are induced and maintained by the (endfeet of) astrocytes (see Chapters 8 and 9), surrounding the BCEC [9, 15], as well as by neuronal endings, which can directly innervate the BCEC [5, 16]. Pericytes also play a role at the blood-brain barrier, as they share the continuous capillary basement membrane with the BCEC. Pericytes cover about 20–30% of the cerebral capillary surface, so they do not really constitute a physical barrier for the movement of solutes across the BCEC. However, their phagocytotic activity does form an additional blood-brain barrier property [8, 17]. Because of these complex interactions between cell types, as well as the dynamic regulation of the blood-brain barrier properties (e.g. receptor expression, formation of tight junctions) this barrier is considered to be an organ protecting the brain [18]. Potential neurotoxic endogenous and exogenous compounds are excluded from the brain by the blood-brain barrier. In addition, the blood-brain barrier maintains ion homeostasis and transports essential nutrients, such as glucose, amino acids, purines, nucleosides, peptides and proteins [4, 6]. There are several influx mechanisms which can be divided into active or passive blood-brain barrier transport mechanisms. Passive diffusion depends on lipophilicity and molecular weight [7]. Furthermore, the ability of a compound to form hydrogen bonds limits its diffusion through the blood-brain barrier [19]. In general, Lipinski’s “rule of five” and the Abraham’s equation can be used to predict the passive transport of a drug molecule across the blood-brain barrier (see Chapters 18 and 20) [20, 21]. Transport of hydrophilic compounds via the paracellular route is limited, while lipophilic drugs smaller than 400–600 Da may freely enter the brain via the transcellular route. Other blood-brain barrier transport systems can be divided into adsorptive-, carrier- or receptor-mediated uptake/transcytosis. Adsorptive-mediated transcytosis is initiated by the binding of polycationic substances (such as most cell-penetrating peptides) to negative charges on the plasma membrane [22, 23]. This process does not involve specific plasma membrane receptors. Upon binding of the cationic compound to the plasma membrane, endocytosis occurs, followed by the formation of endosomes. Indeed, several drugs have been described to enter the brain via this mechanism [24, 25]. However, vesicular transport is actively down-regulated in the blood-brain barrier to protect the brain from nonspecific exposure to polycationic compounds. Therefore, forcing drugs to enter the brain by adsorptive-mediated transcytosis may go against the neuroprotective barrier function, as was recently illustrated for anionic and cationic nanoparticles, which were shown to disrupt the blood-brain barrier [26]. Carrier-mediated transcytosis is used for the transcytosis of nutrients, such as glucose, amino acids and purine bases [27–29]. At least eight different nutrient transport systems have been identified, which each transport a group of nutrients of the same structure. Carrier-mediated transcytosis is substrate-selective and the transport rate is dependent on the degree of occupation of the carrier [29]. In addition, carriers stay in the membrane of the cell, which means that the size of the drugs that can be transported must closely mimic the endogenous ligand if they will be taken up and transported into the brain.

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The focus of this chapter is on receptor-mediated transcytosis by the transferrin receptor [31, 32], the insulin receptor [30] and the transporters for low-density lipoprotein [33], leptin [34] and insulin-like growth factors [35]. These systems enable larger molecules, such as peptides, proteins and genes to specifically enter the brain. Besides many influx mechanisms, several efflux mechanisms exist at the blood-brain barrier as well. The best known is P-glycoprotein (Pgp). Pgp is a transmembrane protein commonly regarded to be located at the apical membrane of the BCEC, although some recent publications describe (conditional) Pgp expression on astrocytes and astrocyte endfeet as well [36]. It has a high affinity for a wide range of compounds, including cytotoxic anticancer drugs, antibiotics, hormones and HIV protease inhibitors [37]. Other multidrug resistance (MDR) efflux mechanisms at the blood-brain barrier include the MDR-related protein (MRP), such as MRP1, -2, -5 and -6 [38]. In addition, many other transporters are present at the blood-brain barrier, such as the organic anion transporter (influx, efflux), the organic cation transport system (influx) and the nucleoside transporter system (influx) [18, 39]. In conclusion, research over the years has shown that the blood-brain barrier is a dynamic organ, which combines restricted diffusion to the brain for endogenous and exogenous compounds with specialized transport mechanisms for essential nutrients.

21.3 Drug Delivery and Targeting Strategies to the Brain

Drug delivery to the brain can be achieved via several methods, including invasive, pharmaco-chemical or physiological strategies. Invasive brain drug delivery strategies, such as direct intracerebral injections of, e.g., slow-release products allow only for local delivery (Fig. 21.2 B). This may be effective for drug delivery to localized brain tumors, but not for the administration of drugs against more widespread diseases. Another invasive method is intracerebroventricular (ICV) or intrathecal drug infusion, in which a drug is directly injected into the CSF. However, the CSF is completely drained into the venous circulation and drugs still have to cross the ependymal brain-CSF barrier. As a result, the infused drug has minimal access to the parenchyma by diffusion (Fig. 21.2 C) [40]. In general, invasive strategies are not effective for drug delivery to the whole brain, but only to a localized part of the brain. The advantage of the vascular route is the widespread diffusion of the infused drug across the whole brain [11]. This can be explained by the large surface area of the human blood-brain barrier (approx. 20 m2). In addition, approximately each neuron has its own brain capillary for oxygen supply as well as the supply of other nutrients (see also Fig. 21.2 A). In fact, every cubic centimeter of cortex comprises the amazing sum of 1 km of blood vessel. This means that the vascular route is a very promising one for drug targeting and delivery to the brain. However, drug delivery through blood-brain barrier disruption by osmotic im-

21.3 Drug Delivery and Targeting Strategies to the Brain

Fig. 21.2 Drug delivery via the vascular route will enable widespread distribution of the drug to each single neuron within the brain. (A) Scanning electron micrograph of a vascular cast of a mouse brain [97]. a = artery; v = vein. Note: the bar indicates 25 lm, which is about the size of a single neuron. (Reprinted with permission from Lippincott Williams & Wilkins). (B) Minimal diffusion of [125I]-nerve growth factor (NGF) after intracerebral implantation

of a biodegradable polymer [98]. Note: the bar indicates 2.5 mm, which is the size of the implant. (Reprinted with permission from Elsevier). (C) ICV injection of [125I]-brain-derived neurotrophic factor (BDNF) [99]. Note that the neurotrophin does not distribute into the brain beyond the ipsilateral ependymal surface. (Reprinted with permission from Elsevier).

balance or vaso-active compounds, though effective in reaching the entire brain, has the disadvantage that neurons may be damaged permanently due to unwanted blood components entering the brain [41]. In contrast, physiological drug delivery strategies rather aim to use endogenous transport mechanisms at the blood-brain barrier, such as carrier- or receptor-mediated transcytosis. The focus of this chapter is on receptor-mediated transcytosis technologies that have successfully been employed to target drugs to the brain.

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21.4 Receptor-Mediated Drug Delivery to the Brain

In general receptor-mediated transcytosis occurs in three steps: receptormediated endocytosis of the compound at the luminal (blood) side, movement through the endothelial cytoplasm and exocytosis of the drug at the abluminal (brain) side of the brain capillary endothelium [8]. Upon receptor-ligand internalization, clathrin-coated vesicles are formed, which are approximately 120 nm in diameter [42, 43]. Therefore, receptor-mediated transcytosis allows the specific delivery of larger drug molecules or drug carrying particles (like liposomes or nanoparticles) to the brain.

21.5 Transferrin Receptor

The most widely characterized receptor-mediated transcytosis system for the targeting of drugs to the brain is the transferrin receptor (TfR). The TfR is a transmembrane glycoprotein consisting of two 90-kDa subunits. A disulfide bridge links these subunits and each subunit can bind one transferrin molecule [31]. The TfR is expressed mainly on hepatocytes, erythrocytes, intestinal cells, monocytes and endothelial cells of the blood-brain barrier [44, 45]. Furthermore, in the brain the TfR is expressed on choroid plexus epithelial cells and neurons [31]. The TfR mediates cellular uptake of iron bound to transferrin. Drug targeting to the TfR can be achieved either by using the endogenous ligand, transferrin or by using an antibody directed against the TfR (OX-26 antirat TfR). Each of these targeting vectors has its advantages and disadvantages. It was shown that transferrin conjugated to horseradish peroxidase (HRP) was very efficiently taken up by blood-brain barrier endothelial cells in vitro (Fig. 21.3) [46]. However, for the TfR, in vivo application is limited due to high endogenous concentrations of transferrin in plasma and the likely overdosing with iron when one tries to displace the endogenous transferrin with exogenously applied transferrin-containing systems. Nevertheless, recent studies in our group have shown that liposomes tagged with transferrin are suitable for drug delivery to blood-brain barrier endothelial cells in vitro, even in the presence of serum (Visser et al., submitted). OX-26 does not bind to the transferrinbinding site and is therefore not displaced by endogenous transferrin. The TfR is responsible for iron transport to the brain. So far, the intracellular trafficking of transferrin and OX-26 upon internalization via the TfR has not yet been elucidated. Some literature reports suggest transcytosis of transferrin across the BCEC, while others claim endocytosis of transferrin, followed by an intracellular release of iron and a subsequent return of apo-transferrin to the apical side of the BCEC [32, 47, 48]. Moos and Morgan [31] have shown that the transcytosis of iron exceeds the transcytosis of transferrin across the blood-brain barrier, supporting the second theory. Furthermore, these authors have proposed

21.5 Transferrin Receptor

Fig. 21.3 Association of Tf-HRP (3 lg ml–1, 2 h, 37 8C) was reduced by a 500-fold excess of nonconjugated Tf, but not by an equal concentration of BSA. Total association of BSA-HRP (3 lg ml–1, 2 h, 37 8C) was approximately 50% of the association of Tf-HRP and was inhibited by both Tf and BSA. ** P < 0.01, Tf-HRP + Tf, or total BSA-HRP vs total Tf-HRP. ## P < 0.01, BSA-HRP + Tf or BSA-HRP + BSA vs total BSA-HRP, one-way ANOVA. (From [46], with permission from Elsevier).

a new theory in which the TfR-transferrin complex is transcytosed to the basolateral side of the BCEC, where transferrin remains bound to the TfR, but iron is released into the brain extracellular fluid [49]. Subsequently, apo-transferrin bound to the TfR will recycle back to the apical side of the blood-brain barrier. This theory is supported by data from Zhang and Pardridge [47], who found a 3.5-fold faster efflux from brain to blood of apo-transferrin than holo-transferrin. In addition, Deane et al. [50] recently illustrated that free iron is also rapidly taken up by brain capillaries and subsequently released into the brain extracellular fluid and CSF at controlled moderate to slow rates. The mechanism of transcytosis of OX-26 is not yet fully elucidated either. Pardridge and colleagues have shown efficient drug targeting and delivery to the brain in vivo by applying OX-26, e.g. [32, 51–53]. In contrast, Broadwell et al. [54] have shown that both transferrin and OX-26 are able to cross the bloodbrain barrier, but that the transcytosis of transferrin is more efficient. Furthermore, Moos and Morgan [55] have shown that OX-26 mainly accumulates in the BCEC and not in the postcapillary compartment. In addition, iron deficiency

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did not increase OX-26 uptake in rats. Both our data and literature reports show that iron deficiency causes an increase in TfR expression [44, 56, 57]. Therefore, it is expected that the uptake of OX-26 would also increase. The data by Moos and Morgan [55] suggest that OX-26 transcytosis might result from a high-affinity accumulation by the BCEC, followed by a nonspecific exocytosis at the basolateral side of the BCEC. In addition, these authors found a periventricular localisation of OX-26, which suggests that OX-26 probably is also transported across the blood-CSF barrier. Although the mechanism of transcytosis of transferrin and OX-26 may not yet be fully elucidated, it is important to realize that drug delivery to the brain via the TfR is efficient. By these means, vasoactive intestinal peptide (VIP), brain derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), peptide nucleic acids and pegylated immunoliposomes containing plasmid DNA encoding beta-galactosidase, tyrosine hydroxylase and short hairpin RNAs have all been made available to the brain [58]. However, OX-26 is an antibody against the rat TfR and does not bind to the human TfR, making it impossible to transfer this technology to the clinic. Moreover, rat antibodies cause immunogenic reactions in humans, unless they are humanized. The preparation of humanized or chimeric antibodies is difficult and in some cases may lead to a loss of affinity for the target receptor [59]. In addition, one can argue that the administration of antibodies directed against such an important uptake mechanism involved in iron homeostasis poses a risk for human application. Still, preferably, a targeting vector directed to the TfR would be small, nonimmunogenic and should initialize internalization of the TfR upon binding. Xu et al. [60] used a single-chain antibody Fv fragment against the human TfR, which was tagged with a lipid anchor for insertion into a liposomal bilayer. The molecular weight of this antibody fragment, including the lipid anchor was approximately 30 kDa. In addition, Lee et al. [61] used a phage display technique to find small peptide ligands for the human TfR. They obtained a 7- and a 12-mer peptide that bind to a different binding site than transferrin and are intenalized by the TfR. Although these small peptides can also exert immunogenic reactions in humans, they are promising ligands for drug targeting to the human TfR on the blood-brain barrier.

21.6 Insulin Receptor

Another widely characterized, classic, receptor-mediated transcytosis system for the targeting of drugs to the brain is the insulin receptor. Again, just as for the TfR system, Pardridge and colleagues have predominantly documented the use of the insulin receptor for the targeted delivery of drugs to the brain. The insulin receptor is a large 300-kDa protein and is a heterotetramer of two extracellular alpha and two transmembrane beta subunits. Each beta chain con-

21.7 LRP1 and LRP2 Receptors

tains a tyrosine kinase activity in its cytosolic extension. The alpha and beta subunits are coded by a single gene and are joined by disulfide bonds to form a cylindrical structure. Primarily, insulin binds and changes the shape of the receptor to form a tunnel, to allow entry of molecules such as glucose into the cells. The insulin receptor is a tyrosine kinase receptor and induces a complex cellular response by phosphorylating proteins on their tyrosine residues. The binding of a single insulin molecule into a pocket created by the two alpha chains effects a conformational change in the insulin receptor so that the beta chains approximate one another and carry out transphosphorylation on tyrosine residues. This autophosphorylation is necessary for the receptor to internalize into endosomes. The endosomal system has been shown to be a site where insulin signaling is regulated, but where the degradation of endosomal insulin also occurs. Most of the insulin is degraded, but less so in endothelial cells [62], whereas the receptors are largely recycled to the cell surface. Endocytosis is not necessary for insulin action, but probably is important for removing insulin from the cell surface so that the target cell for insulin responds to the hormone in a time-limited fashion. This endocytotic mechanism of the insulin receptor has been exploited for the targeting of drugs to the brain. As for transferrin, the in vivo application of insulin as the carrier protein is limited, mainly due to the high insulin concentrations needed and the resulting lethal overdosing with insulin. Therefore, drug or gene delivery to, for instance, rhesus monkeys is performed with the mouse 83–14 mAb that binds to the exofacial epitope on the alpha subunit of the human insulin receptor. The mAb has a blood-brain barrier permeability surface area (PS) product in the primate that is 9-fold greater than mouse mAbs to the human TfR [63]. By using this mAb, Pardridge and coworkers have successfully made radiolabeled amyloid-b peptide1–40 (Ab 1–40), serving as a diagnostic probe for Alzheimer’s disease, and pegylated immunoliposomes containing plasmid DNA encoding b-galactosidase available to the brain of primates [58]. Unfortunately, the 83–14 mAb cannot be used in humans owing to immunogenic reactions to this mouse protein. However, genetically engineered, effective, forms of the mAb have now been produced, which may allow for drug and gene delivery to the human brain [64]. Still, one can argue that the administration of antibodies directed against such an important mechanism involved in glucose homeostasis poses a risk for human application.

21.7 LRP1 and LRP2 Receptors

During the past few years, the low-density lipoprotein receptor related protein-1 (LRP1) and LRP2 (also known as megalin or glycoprotein 330) receptors have been exploited to target drugs to the brain in a fashion similar to that used for the transferrin and insulin receptors. Both LRP1 and LRP2 receptors belong to the structurally closely related cell surface LDL receptor gene family. Both recep-

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tors are multifunctional, multiligand scavenger and signaling receptors. A large number of substrates are shared between the two receptors, like lipoprotein lipase (LPL), a2-macroglobulin (a2M), receptor-associated protein (RAP), lactoferrin, tissue- and urokinase-type plasminogen activator (tPA/uPA), plasminogen activator inhibitor (PAI-1) and tPA/uPA : PAI-1 complexes. More specific ligands for the LRP1 receptor are described to be, e.g., melanotransferrin (or P97), thrombospondin 1 and 2, hepatic lipase, factor VIIa/tissue factor pathway inhibitor (TFPI), factor VIIIa, factor IXa, Ab1–40, amyloid-b precursor protein (APP), C1 inhibitor, complement C3, apolipoproteinE (apoE), Pseudomonas exotoxin A, HIV-1 Tat protein, rhinovirus, matrix metalloproteinase 9 (MMP-9), MMP-13 (collagenase-3), sphingolipid activator protein (SAP), pregnancy zone protein, antithrombin III, heparin cofactor II, a1-antitrypsin, heat-shock protein 96 (HSP-96) and platelet-derived growth factor (PDGF, mainly involved in signaling) [65–69], where apolipoproteinJ (apoJ, or clusterin), Ab bound to apoJ and apoE, aprotinin and very low-density lipoprotein (VLDL) are more specific for the LRP2 receptor [70, 71]. The group of Richard Béliveau first reported that melanotransferrin/P97 was actively transcytosed across the blood-brain barrier and suggested that this was mediated by the LRP1 receptor [69]. Melanotransferrin is a membrane-bound transferrin homolog that can also exist in a soluble form and is highly expressed on melanoma cells compared to normal melanocytes. Intravenously applied melanotransferrin delivers the majority of its bound iron to the liver and kidney, where only a small part is taken up by the brain [72]. After conjugation to melanotransferrin, the group of Béliveau was able to successfully deliver doxorubicin to brain tumors in animal studies [73, 74]. This melanotransferrinmediated drug targeting technology (now designated NeuroTrans) is currently being further developed by BioMarin Pharmaceuticals Inc. (Novato, Calif., USA) for the delivery of enzyme replacement therapies to the brain. Interestingly, together with researchers from BioMarin, Pan et al. [75] recently reported on the efficient transfer of RAP across the blood-brain barrier by means of the LRP1/ LRP2 receptors, suggesting the discovery of a novel means of protein-based drug delivery to the brain. RAP is a 39-kDa protein that functions as a specialized endoplasmic reticulum chaperone assisting in the folding and trafficking of members of the LDL receptor family. In yet unpublished results, the group of Béliveau have now filed a patent application on the use of the LRP2-specific ligand aprotinin, and more specifically on functional derivatives thereof (e.g. angio-pep1), thereby providing a noninvasive and flexible method and a carrier for transporting a compound or drug across the blood-brain barrier [76]. Aprotinin (Trasylol) is known as a potent inhibitor of serine proteases such as trypsin, plasmin, tissue and plasma kallikrein and is the only pharmacologic treatment approved by the United States Food and Drug Administration to reduce blood transfusion in coronary artery bypass grafting [77]. In addition to being a tumor marker protein, melanotransferrin is also associated with brain lesions in Alzheimer’s disease and is a potential marker of the disorder [78]. In addition, the proposed receptor for melanotransferrin, LRP1,

21.8 Diphtheria Toxin Receptor

has genetically been linked to Alzheimer’s disease and may influence APP processing and metabolism and Ab uptake by neurons through a2M (a2M is one of the Ab carrier proteins next to, e.g., apoE, apoJ, transthyretin, albumin) [70, 79]. Furthermore, a close relationship with RAGE (receptor for advanced glycation end products), in shuttling Ab across the blood-brain barrier has been described [70, 79]. In addition, the LRP2 receptor has also been described to mediate the uptake of Ab complexed to apoJ and apoE across the blood-brain barrier [71, 80, 81]. This complex interaction with Alzheimer’s disease makes it difficult to predict the safety of the use of the LRP1/LRP2 receptors for the targeting of drugs to the brain in human application. This is especially so when the complex signaling function of these receptors is included in the assessment (including e.g. control of permeability of the blood-brain barrier, vascular tone, expression of MMPs) [66], as well as the fact that both the receptors are critically involved in the coagulation-fibrinolysis system. On top of which, melanotransferrin is also reported to be directly involved in the activation of plasminogen [82]; and high plasma concentrations of melanotransferrin are needed to deliver drugs to the brain, resulting perhaps in dose limitations because of the high iron load in the body. The same line of reasoning for the interactions at the level of the uptake receptors may apply to the use of RAP and aprotinin (derivatives). However, the latter has already been successfully applied to humans usually without severe side-effects, indeed making the peptide derivatives potentially safe drug carriers. As for RAP, though, no results on the efficacy or capacity of the aprotinin peptides as carrier for drugs have been made available yet.

21.8 Diphtheria Toxin Receptor

Recently, our group identified a novel human-applicable carrier protein (known as CRM197) for the targeted delivery of conjugated proteins across the bloodbrain barrier [83, 84]. Uniquely, CRM197 has already been used as a safe and effective carrier protein in human vaccines for a long time [85] and recently also as a systemically active therapeutic protein in anti-cancer trials [86]. This has resulted in a large body of prior knowledge on the carrier protein, including its transport receptor and mechanism of action, receptor binding domain, conjugation and manufacturing process and its kinetic and safety profiles, both in animals and humans. CRM197 delivers drugs across the blood-brain barrier by the well characterized, safe and effective mechanism called receptor-mediated transcytosis. From the literature, it was already known that CRM197 uses the membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor (HB-EGF) as its transport receptor [87]. This precursor is also known as the diphtheria toxin receptor (DTR). In fact, CRM197 is a nontoxic mutant of diphtheria toxin. Membrane-bound HB-EGF is constitutively expressed on the blood-brain barrier, neurons and glial cells [88]. Moreover, HB-EGF expression

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is strongly up-regulated on the cerebral blood vessels by for instance ischemic stroke and in gliomas [89, 90], which may lead to a site-selective improvement of the therapeutic efficacy of the targeted drugs in the brain. By means of the dynamic cell culture model of the blood-brain barrier [91], our group was able to demonstrate the functional expression of the DTR, safety of the CRM197 carrier protein and specific transport efficacy of CRM197 carrier protein conjugates to a 40-kDa enzyme (HRP, serving as a “model” protein drug) and DTR-targeted pegylated liposomes containing HRP. In addition, the in vivo “proof of principle” with this novel brain drug-targeting technology was demonstrated by the specific brain uptake of DTR-targeted HRP in guinea pigs [83, 84]. Although HB-EGF is expressed in species including human, monkey, rat and mouse with a similar tissue distribution, only rat and mouse are resistant to diphtheria toxin because of an amino acid substitution in the receptor-binding domain on HB-EGF that reduces the binding of diphtheria toxin to rodent HBEGF [92]. Fortunately, a transgenic mouse (conditionally) expressing the human DTR was recently generated by Dr. Cha and coworkers [93], allowing the specific study of the brain drug delivery technology in mice as well. Another known complication of the bacterial CRM197 protein is that neutralizing antibodies against diphtheria toxin may develop or already be present in the serum of the recipient because of earlier vaccinations, thereby reducing the efficacy of the drug delivery system. There are, however, several lines of evidence that such an immune response to CRM197 can occur, but, most importantly, that it is not really a problem in the clinic, at least not for the treatment of acute indications. In fact, the clinical studies performed by Buzzi et al. [86] indicate that pre-existing levels of neutralizing antibodies were rather decreased 30 days after repeated treatment with CRM197. Overall, the DTR seems to be a human-applicable, safe and effective uptake receptor for the targeting of drugs to the brain, especially since CRM197 is already safely applied to humans (illustrating that binding to HB-EGF per se does not result in serious side-effects), where other carrier systems involve potential safety hazards in human application. However, even though specific brain uptake of DTR-targeted enzyme is already established in guinea pigs, the technology now awaits further in vivo validation in terms of kinetics of brain distribution and efficacy of targeted drugs in relevant disease models of the CNS.

21.9 Conclusions

The multibillion dollar CNS drug market (US $ 58 ´ 1012 in 2003) is rapidly growing (14% annually) and bears significant unmet medical needs. For this reason, the CNS drug market is considered as the most promising segment for the whole drug industry. However, many (biopharmaceutical) drugs are currently not available to the brain because they are not able to enter the brain due

21.9 Conclusions

to the presence of the blood-brain barrier. The stubborn assumption that the breakdown of the blood-brain barrier (which occurs in the course of most CNS disorders) will eventually solve the problem has been proven wrong by those from industry and academia who have already tried. Particularly, either it breaks down after the therapeutic time-window or the drug is still not able to cross the “leaky” barrier in sufficient quantities to be effective. Just to give one devastating example, the brain is a sanctuary site for the metastasis of breast tumors normally curable by therapeutic anticancer antibodies that are not able to cross the blood-brain barrier, making this type of therapy overall ineffective for these patients [94]. Brain drug-targeting technology may solve this problem. So perhaps the most shocking conclusion from this chapter is that, to date, there are still no drugs in the clinic, let alone on the market, that employ brain drug-targeting technologies. Clearly, despite considerable efforts, small molecules have thus far not been able to solve the unmet needs. The characteristics required for sufficient brain penetration of small molecules are usually not in line with the safety requirements of the compounds. For that reason, more can be expected from the emerging field of biopharmaceutical drugs. Unlike small molecules, however, biopharmaceutical drugs are unlikely candidates for chemical modifications to enhance their permeability across the blood-brain barrier. Only invasive and harmful technologies to patients, like direct and local stereotactic injections, intrathecal infusions and blood-brain barrier disruption, are currently being evaluated in clinical settings. Because of the severe neurological consequences of these techniques, however, these are only allowed to be applied in selected life-threatening diseases. Moreover, these technologies are far from effective in delivering drugs throughout the whole brain. Since almost every neuron is perfused by its own capillary, the most effective way of delivering biopharmaceutical drugs is achieved by targeting to internalizing (uptake) transport receptors on these capillaries. In this chapter, we have highlighted the possibilities and limitations of the human applicability of the transferrin-internalizing (uptake) transport receptor, including the insulin receptor, LRP1 receptor, LRP2 receptor and diphtheria toxin receptor. So what will it take to get these systems into the clinic? Clearly the transferrin receptor-targeted system has been best validated in terms of the concept of targeted drug delivery to the brain, but not yet for the recently discovered human-applicable peptide carriers. The insulin receptor-targeted system is the closest to human validation because of the successful “proof of concept” studies in primates. Moreover, the potential safety issue of antibodies directed against insulin receptors has, to our knowledge, not yet been properly addressed. In addition, the on/off phenomenon of antibody-receptor binding/release seems to be strongly in favor of binding which may result in cellular accumulation and limited transcytosis [95]. In situ perfusion studies in rats demonstrated that OX-26 and OX-26-conjugated liposomes were selectively distributed to BCECs, leaving the choroid plexus epithelium, neurons and glia unlabeled [96]. However, this does not mean that a liposomal cargo could not be transported into the brain.

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Further, the targeted delivery of drugs to the brain through the LRP1 receptor may still face considerable difficulties in evading possible interactions with both the numerous other ligands of the receptor as well as the established signaling function of the receptor. For these reasons, the use of uptake receptors that already have an exogenously applied ligand for the receptor being applied to humans, like for the DTR (i.e. CRM197), may benefit considerably from the derived safety data coming from other such applications. In fact, as soon as the beneficial effects of the CRM197 carrier protein conjugated to already-marketed drugs are observed in relevant animal disease models, the preparation of an IND package is warranted for certain life-threatening CNS disorders. In conclusion, what is essentially needed to get brain-targeted delivery systems into the clinic without further delay, is the involvement of companies with drugs already on the market that need their drug to be made available to the brain as well. Combining such drugs to brain drug targeting and/or delivery technologies that individually already have regulatory approval for other indications, may provide the optimal building blocks to obtain the clinical success of brain-targeted delivery systems that so many patients have been waiting for. In addition, these delivery systems can then be subsequently applied to target biopharmaceutical drugs (such as enzymes, siRNAs, genes) to the brain. Furthermore, drug delivery technologies combined with brain-targeting systems may also help to validate the many newly discovered drug targets in a much better way. We envision that a relevant drug target is first validated in animal disease models with the best possible class of lead compounds that usually do not cross the blood-brain barrier on their own, before chemical modifications are introduced to a preferred compound to make it available to the brain.

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Part VI Vascular Perfusion


523

22 Blood-Brain Transfer and Metabolism of Oxygen Albert Gjedde

22.1 Introduction

Oxygen is essential to brain function. Disruption of oxygen delivery to brain results in complete cessation of functional activity in the human brain in 6 s (Hansen 1984, 1985). The reason for this dramatic effect is not known with certainty. Increased potassium conductance in neuronal membranes plays a role, but the effect also suggests that oxygen reserves in brain tissue are very low in relation to the steady-state turnover, and it implies that oxygen turnover is intimately related to the mechanisms that subserve consciousness, because the effect cannot be explained by depletion of intermediary metabolites. The delivery of oxygen to brain tissue differs in major respects from the delivery of oxygen to other tissues. In the classic description of Krogh (1919), the standard capillary bed is a system of parallel tubes serving uniform cylinders of tissue (known as Krogh cylinders). This simple arrangement yields a quantitative expression of oxygen delivery to the tissue, suggestive of specific mechanisms of regulation. However, in brain tissue, the vascular bed differs from the vascular beds of other tissues in two important ways. Both the absence of the recruitment of capillaries to neurons in a state of activation and the general principle of the topographic arrangement of the vessels are features unlike those of other vascular beds, but the importance of these differences has eluded students of the vascular physiology of the brain. The absence of recruitment suggests that the work of the brain, measured as energy turnover, varies little over time; and the random arrangement of capillaries suggests that the geometry of the vascular network is fixed competitively by the work of the nerve cells, rather than by the need to make frequent, orderly and substantive changes to the diffusion capacity, as in skeletal and cardiac muscles. If the geometry is always the same, there is no need for any particular order. Thus, in brain, the vascular bed is so poorly organized that no single geometric description has general validity and hence no single prediction of oxygen Blood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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22 Blood-Brain Transfer and Metabolism of Oxygen

distribution in the tissue is possible (Wang and Bassingthwaighte 2001). To predict the general properties of oxygen delivery and consumption, the remaining valid approach is the averaging of properties across large volumes of tissue, on the strength of observations that have wide applicability, always with the provision that local and regional properties may differ significantly from the global average. Only two claims of the capillary bed in brain appear to be indisputable, i.e., that the capillaries have multiple arterial sources but a common venous terminus and that the density of capillaries is proportional to the average regional rate of metabolism at the steady-state (also known as the “default” state). From these claims arises a revised distribution of oxygen in brain tissue that is different from the classic distribution in Krogh cylinders. The revision is made possible by the simple realization, first introduced by Erwin R. Weibel (1984) in “The Pathway for Oxygen”, that every segment of the capillary bed exists to supply an equivalent amount of brain tissue with oxygen, i.e., that every fraction of the tissue is served by commensurate fractions of a capillary bed, having the appropriate oxygen diffusibility and accounting for equivalent fractions of the total oxygen consumption. This approach considers the random delivery of oxygen molecules from the source in arbitrarily distributed capillaries to the sink in equally arbitrarily distributed mitochondria. The only discernable regulation is the need to make enough oxygen molecules available to the cells served by the capillaries. The approach assumes that suitable mechanisms originally served to place the capillary bed and the cells in relation to each other in such a way that this requirement would be fulfilled. It is an intriguing possibility that the astrocytic projections, in addition to other services, could provide a structural restraint on the anatomic relationship of capillaries and neurons, established to achieve the optimal match of randomly placed capillaries and cells. The current approach is not limited to capillaries per se. It is suspected that oxygen exchange with the tissue may begin in arteriolar microvessels (Duling and Berne 1970). For the purposes of this analysis, arteriolar microvessels are therefore part of the effective capillary bed. The variable organization can be reduced to compartments of predictable behavior by making the simple assumption that the structure of the vascular bed evolved to satisfy the existing needs for oxygen, in contrast to the more traditional view that the needs for oxygen can be deduced from a specific structure of the vascular bed. It turns out that the resulting temporal and anatomical averages of the random processes of oxygen delivery achieve an appearance of orderliness that is illusory.

22.2 Blood-Brain Transfer of Oxygen

22.2 Blood-Brain Transfer of Oxygen 22.2.1 Capillary Model of Oxygen Transfer

Capillary models of blood-brain transfer focus on the distribution of molecules inside microvessels. In the case of molecular oxygen, the extraction from the capillary to the tissue establishes an oxygen profile in the capillary bed that ranges from the tension associated with the more or less fully saturated hemoglobin at the arterial inlet to the tension of the more or less fully desaturated hemoglobin at the venous outlet. Between these extremes, the tension depends on the extraction of oxygen from the network of capillaries serving the tissue. The discussion above leads to the conclusion that it is impossible to make a specific prediction of the profile in individual capillaries but it is possible to look at the ensemble of capillaries as a unit that provides the tissue with the oxygen it needs. This simple assumption leads to a solution that is close, but not identical, to the general claim that the oxygen tension declines monoexponentially from the arterial inlet to the venous outlet. The solution is obtained by letting the oxygen extraction from the blood flowing in the general direction from the arterial inlet to the venous outlet be proportional to the fraction of the capillary bed cumulatively served by the blood flow. The fractional segment of the capillary bed is denoted by a variable z that ranges from 0 to 1. In every segment, an equivalent amount of oxygen is extracted by the cells served by their respective segments of the capillary bed: EO2 z

JO2 z zJO2 art FCO2 FCOart2

22:1

where the oxygen extraction fraction is EO2 z; JO2 z is the segmental rate of cerebral metabolism of oxygen (CMRO2) at the segment z of the capillary bed in question, JO2 is the measured CMRO2 value per unit mass or volume of the brain region served by this particular capillary bed, F is the measured blood

Table 22.1 Average properties of oxygen compartments in human brain. (Modified from Gjedde et al. 2005). Variable

Unit

Arterial

Capillary

Venous

CO2 PO2 P50 h SO2

mM mmHg mmHg

8.5 114.0 26.0 2.84 0.985

7.0 53.0 35.0 3.5 0.81

5.3 40.0 35.0 3.5 0.61

Ratio

Tissue

Mitochondrial

20–30

8.5 0.5 0.94

525

526


Fig. 22.1 Oxygen tension, extraction fraction, and oxygen diffusibility as function of axial segmentation of cortical capillary bed (z direction), calculated for baseline condition of human brain. Abscissa: axial segments of capillary bed (z). Ordinate: capillary oxygen extraction fraction EO2 z, cap tension PO2 z, and diffusibility [L(z)] as functions of axial segment. The variables were calculated with Eqs. (22.7) to (22.9) under normal conditions of whole human

brain, as listed in Tables 22.1 and 22.2: normal CMRO2 (141 lmol hg–1 min–1 at the baseline), normal blood flow (F, 43 ml hg–1 min–1), normal arterial oxygen concentration cap (8.7 mM), normal P50 , the half-saturation tension of oxygen (35 mmHg when corrected for the Bohr shift), and normal h, the Hill coefficient (3.5 when corrected for Bohr shift). cap PO2 0 was assumed to equal the arterial oxygen tension POart2 (114 mmHg).

flow to the region, and COart2 is the measured arterial oxygen concentration at the arterial inlet. Standard properties of arterial blood are given in Table 22.1. The extraction of oxygen from brain capillaries causes the capillary oxygen tension to decline from the arterial oxygen tension at the arterial end of the capillary bed to the venous oxygen tension at the venous end. The decline of the oxygen tension as a function of z is determined by the oxygen remaining after the extraction. The loss of oxygen establishes a declining oxygen saturation of hemoglobin according to the formula: cap

SO2 z Sart O2 1

EO2 z

22:2 cap

where Sart O2 is the saturation of hemoglobin in arterial blood and SO2 is the saturation in capillary blood at the segmental position z. The amount of oxygen remaining in the capillary bed also satisfies the Hill equation of the relationship between oxygen tension and hemoglobin saturation:

22.2 Blood-Brain Transfer of Oxygen cap

SO2 z

1

1

cap P50 z cap PO z 2

hz

22:3

cap

where PO2 z is the oxygen tension as a function of position in the capillary, cap P50 z is the half-saturation tension of oxygen at the position z, and h(z) is the value of the Hill coefficient at that position. The Hill equation then is solved for the capillary oxygen tension associated with the particular extraction fraction: s Sart EO2 z cap cap hz O2 1 PO2 z P50 z 22:4 1 EO2 z 1 Sart O2 which, on the assumption that the saturation of hemoglobin in arterial blood is negligibly different from unity, reduces to the expression: s 1 cap cap hz 22:5 PO2 z P50 z 1 EO2 z cap

where PO2 z is the pressure head of oxygen diffusion when arterial oxygen saturation is 100%.1) The third determinant of oxygen delivery is the oxygen diffusibility, which depends on the diffusion coefficient, the solubility, and the position of the oxygen sink or sinks. The oxygen diffusibility is given by the ratio between the oxygen flux and the difference between the pressures of oxygen in the z segment of the capillary bed and in the mitochondria of the tissue segment served by the segment of capillary bed: Lz

JO2 cap mit z PO2 z PO 2

22:6

where the oxygen diffusibility L(z) is a function of the value of the segmental z is the operative mitochondrial oxygen tension at the site, position z and POmit 2 which a priori is not known with certainty in brain tissue, although attempts have been made to calculate the value (Gjedde et al. 2005).

1) As an alternative to the assumption of unity

denominator assumes a value within a fewpercentage points of unity for ordinary extraction fractions at arterial oxygen tensions above 100 mmHg. At lower oxygen tensions, the value of the denominator depends on the position in the capillary bed. Since the physically dissolved oxygen represents no more than 1–2% in norcap P50 z moxia (although less in hypoxia), the physically app P50 z r ven dissolved oxygen “spares” some of the bound hz 1 SO2 in Sart Sven oxygen during extraction. There is indication O2 O2 that the half-saturation tension of arterial blood app which P50 is the half-saturation tension cor- is adjusted in hypoxia to counter the effects of rected for arterial desaturation of hemoglobin the low arterial hemoglobin saturation (Gjedde and Sven O2 is the venous oxygen saturation. The 2002).

for Sart O2 , it is possible to lump the effect of arterial desaturation into an apparent change of the half-saturation constant of hemoglobin, app P50 z. The apparent half-saturation constant is then given by:

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The diffusibility depends on the surface area of the capillary segment. The diffusion gradient also varies with the distribution of mitochondria in the tissue, which is not known with certainty for any specific location and hence must be inferred from consideration of averages across larger regions. If the sinks are placed at the end of the diffusion path, L(z) is an estimate of the diffusion coefficient, corrected for solubility, that reflects the average distance from the z segment to the mitochondria served by the segment. The distinction between even and uneven distribution of metabolic sinks for oxygen will be discussed in greater detail in the subsequent section on tissue oxygen tensions. Equations (22.1), (22.5), and (22.6) can be solved in sequence, beginning with the extraction fraction, as functions of the capillary segment z. The results are shown in Fig. 22.1. The extraction rises linearly as shown in Eq. (22.1); and the capillary oxygen tension declines accordingly as shown in Eq. (22.5). The decline is approximately monoexponential as predicted by conventional capillary models (Crone 1963), but the reason is not per se the simple pressure gradient between capillary and tissue, but is the more complex satisfaction of a constant oxygen delivery, organized as a combination of the linearly increasing extraction fraction and the sigmoidal relationship between oxygen tension and hemoglobin saturation. The diffusibility also rises in response to the needs of the tissue segment served, to maintain the constant flux of oxygen; and the increase is not linear. A nonlinearly rising diffusibility can be achieved in several ways, including a declining distance between adjacent microvessels as well as by branching of vessels.

22.2.2 Compartment Model of Oxygen Transfer

A compartment is an idealization of kinetic relationships in which all concentration gradients are placed at the interfaces among the compartments. Inside the compartments, in contravention of the true nature of gradients, concentrations are considered to be uniform. The linearity of the extraction fraction means that the capillary bed can be reduced to a single microvascular compartment with a cap , derived from the linearly averaged extraction fracuniform oxygen tension, P O2 tion. The linearity of the extraction fraction as function of the capillary segmentation further implies that the entire axial dimension can be projected onto a single transaxial plane at the position z = 0.5, where the extraction fraction is O =2. Provided the arterial sathalf of the totally extracted fraction EO2 0:5 E 2 uration of hemoglobin is close to unity, the weighted average capillary oxygen tension associated with the oxygen present in all segments of the capillary bed (Vafaee and Gjedde 2000) is then: cap cap P P 50 O2

s 2 h 1 O E 2

22:7

22.3 Oxygen in Brain Tissue

where P 50 is the average capillary half-saturation tension of oxygen binding to is the average value of the Hill coefficient in the capillary. hemoglobin2) and h When EO2 z rises linearly, the corresponding weighted average oxygen diffusibility is: cap

cap JO2 L mit P P O2 O2

22:8

mit is the tissue’s minimum oxygen tension, and L is the mean oxygen where P O2 depends on the undiffusibility, equal to Lz at z = 0.5. The magnitude of L mit O , the oxygen tension at cytochrome c oxidase. Equations known value of P 2 by identifying an extraction fraction (22.7) and (22.8) can be used to estimate L at which the oxygen reserve of the mitochondria has been exhausted and calcu by means of Eq. (22.8). It follows from Eq. lating the corresponding value of L (22.8) that the oxygen consumption is: 2

JO2

s 2 cap h 4 L P50 1 ^O E

3 mit 5 P O2

22:9

2

Solutions to these equations are shown in where the average value of h is h. Figs. 22.1 and 22.2 for normal values of human brain.

22.3 Oxygen in Brain Tissue 22.3.1 Cytochrome Oxidation

The solutions to the equations above depend on the magnitude of the mitochondrial oxygen tension in Eqs. (22.8) and (22.9). This tension, together with the affinity of cytochrome c oxidase towards oxygen, the maximum reaction rate of each mitochondrion, and the number of mitochondria in a given segment of tissue, determines the oxygen consumption according to the simplest formulation of the Michaelis-Menten equation (Gnaiger et al. 1995, 1998; Guilivi 1998):

cap

P Sart O2

50 app s 2) As an alternative to the assumption of arterial P which has the value 50

hemoglobin saturation of unity, it is again possible to define an apparent average halfsaturation tension that corrects for the degree of desaturation of hemoglobin that exists prior to the entry into the capillary. In the compartmental model, in agreement with app is given by the expression Eq. (4), P 50

Sven O2 ven Sart S O2 O2 1.02 for an extraction of 40% at an arterial oxygen tension of 114 mmHg (standard). At a tension of 52 mmHg, the denominator has the values 1.16 at an extraction of 40% and 1.12 at 60% extraction. h

2

529

530


Fig. 22.2 Capillary oxygen profiles. Abscissa: axial segment of capillary bed cap (z). Ordinate: capillary oxygen tensions PO2 z as a function of axial segment, calculated as explained in Fig. 22.1 for baseline (CBF 43 ml kg–1 min–1), and two oligemia (low blood flow) states (CBF 26.5 ml hg–1 min–1, 21 ml hg–1 min–1), chosen to be just at and just below the limit of adequate oxygen delivery. Numerical solution of the three equations with the three cap unknowns EO2 z, PO2 z, and x0 (z) yielded the capillary oxygen tension profile for insufficient oxygen supply (see Section 22.5).

JO2

Jmax POmit 2 cyt

mit P50 PO 2 cyt

22:10

where P50 is the apparent half-saturation tension of the oxygen reaction with cyis the mitochondrial oxygen tension, and Jmax is the tochrome oxidase, POmit 2 maximum velocity of the cytochrome oxidase reaction. This equation is applicable to all mitochondria, but it is not known to what extent the magnitudes of each of the parameters of the equation, as well as the resulting oxygen consumption, apply generally to the tissue as a whole. Therefore it is necessary to operate with the concept of an average mitochondrion. In vivo, the affinity is considerably (an order of magnitude) below the inherent affinity observed under in vitro circumstances. The expected saturation of the enzyme therefore is not as pronounced in vivo as in vitro. The standard properties of this enzyme are given in Table 22.1. The actual flux of oxygen in each tissue volume element depends on the regional oxygen consumption, which is often assumed to be the same per unit tis-


sue mass or volume everywhere but may in fact vary considerably from site to site. The flux is the difference between the average rate of oxygen consumption of the tissue segment and the regionally (but not locally) invariant rate of oxygen delivery to each unit of tissue. The mitochondrial oxygen tension in turn links the oxygen consumption with the oxygen delivery. The mitochondrial oxygen tension represents the balance between the diffusion of oxygen to the site of metabolism and the reaction of oxygen with cytochrome c oxidase, according to the simple Michaelis-Menten expression. The local variability of mitochondrial oxygen tension in brain tissue is therefore the key to the understanding of oxygen homeostasis.

22.3.2 Mitochondrial Oxygen Tension 22.3.2.1 Distributed Model of Tissue and Mitochondrial Oxygen In the classic model of nonuniformly distributed oxygen, the mitochondria are evenly scattered in the tissue but the oxygen tension varies spatially as oxygen is consumed. This situation is reminiscent of the conditions existing in the Krogh cylinder, in which the local mitochondrial oxygen tension varies with the distance of the mitochondria from the microvessels, while the oxygen consumption rate is assumed to be the same for each mitochondrion, because affinity or maximum reaction rate or both are adjusted to maintain the same oxygen consumption rate in every tissue element. This situation is consistent with a one-dimensional diffusion equation of the form:

jO2 z; x

KAz

qpO2 z; x 1 qx

xJO2 z

22:11

where jO2 z; x is the flux of oxygen from the capillary segment z to the tissue segment x, JO2 z is the regionally invariant oxygen consumption of each unit of tissue mass or volume, K is Krogh’s diffusion coefficient for a unit surface area, and A(z) is the magnitude of that area. The sites of oxygen consumption generate an extravascularly directed gradient of oxygen tension from the microvascular compartment to the tissue mass or volume fraction x served by the microvascular compartment: dPO2 x dx

1 x JO2 K A

22:12

is the diffusion area of a segmental surface. Integration with respect to where A the tissue mass or volume fraction x yields the pressure as a function of the tissue mass or volume fraction served by this segment of the capillary:

531

532


JO2 PO2 x PO2 1 1 2K A

x2

22:13

where PO2 1 is the minimum oxygen tension in the tissue. For evenly distributed (i.e., equally abundant more and less “distant”) sites of oxygen consumption in the one-dimensional tissue, obtained by projecting the higher dimensions onto the segment at z = 0.5, the relationship between Krogh’s coefficient K and the weighted average oxygen diffusibility is: 2K A L

22:14

for K A yields the weighted average oxygen tension such that substitution of L=2 as a function of the weighted average distance from the capillary: PO2 x P O2

cap

JO2 2x L

x2

22:15

is the weighted average oxygen diffusibility defined in Eq. (22.5), and where L JO2 is the average rate of oxygen consumption of the entire tissue. This solution is shown in Fig. 22.3 for the cases of one normal and two low levels of blood flow to the human cerebral cortex. For x = 1.0, Eq. (22.15) yields the minimum mitochondrial oxygen tension reached at the end of the diffusion path: cap

mit PO2 PO 2

JO2 L

22:16

which is identical to the evenly distributed oxygen tension associated with the linear diffusion case above. Integration and normalization of Eq. (22.15) yields the weighted tissue average of the oxygen tension: cap br P P O2 O2

2JO2 3L

22:17

br O where P is the weighted average tissue tension. 2 Equation (22.15) is in fact a special solution to a more general formulation in which the local mitochondrial oxygen tension varies with the distance from the microvessels, depending on the local oxygen consumption rate, which in turn varies with the oxygen tension according to the Michaelis-Menten equation for cytochrome c oxidase, provided the enzyme has the same affinity and maximum reaction rate everywhere. In that case, the diffusion equation assumes the form:

d2 PO2 x PO x Jmax cyt 2 K A dx 2 P50 PO2 x

! 22:18


Fig. 22.3 Tissue oxygen tensions of distributed tissue model. Abscissa: tissue volume fraction (x). Ordinate: oxygen tension profiles PO2 x as a function of tissue volume fraction for the three cases in Fig. 22.2, using the equations indicated in the figure, identical to Eqs. (22.13) and (22.24) in the text. The normal end-path oxygen tension was assumed to be 8.5 mmHg, as listed in Table 22.1.

which equation in principle is soluble only by numerical means. When cyt PO2 x P50 , the equation reduces to the special case of spatially uniform oxygen consumption discussed above. Whether this happens depends on the tension as well as on the affinity.

22.3.2.2 Compartment Models of Tissue and Mitochondrial Oxygen In the simplest case of a compartment model, the local mitochondrial oxygen tensions are uniformly negligible with respect to the capillary oxygen tension and the oxygen diffusion from the capillary compartment, although they remain significant with respect to the cytochrome c oxidase reaction. In this case, the local oxygen consumption still varies with the negligible tension but has little influence on the delivery of oxygen to the tissue, which is then entirely flowlimited. This is the basis for the original explanation of the apparent uncoupling of blood flow from oxygen consumption during functional activation (Buxton and Frank 1997; Gjedde 1997), which also cannot be sustained in light of the maintenance of normal oxygen consumption rates during substantial flow decreases, for example in hypocapnia (Gjedde et al. 2005). In this case, the oxygen diffusibility is simply the ratio between oxygen consumption and the capillary oxygen tension.

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534


The negligible mitochondrial oxygen tension is in reality a special case of a more general situation in which the local tension is maintained at a constant magnitude with uniform distribution, implying that the majority of mitochondria reside at the same distance from the relevant microvessels and have about the same rate of oxygen consumption. In this case, the oxygen is delivered by the simplest diffusion relation established by the tension difference between the microvessels and the mitochondria. The fixed distance from the relevant microvessels and the mitochondria represents a diffusion barrier, for which the tension at the far end is given by cap mit P P O2 O2

JO2 L

22:19

mit is the tension of the uniformly distributed mitochondrial oxygen. where P O2 The resulting compartmental model is shown in Fig. 22.4. The gradient associated with the interface between the capillary and mitochondrial compartments allows the approximation of an average tissue oxygen diffusibility according to Eq. (22.16). It is of course possible that distributed and compartmental oxygen states coexist in the tissue. The local mitochondrial oxygen tension, affinity, maximum reaction rate, and distance from microvessels may vary unpredictably for all mitochondria, the density of which may also vary to maintain the appropriate oxygen consumption for each tissue element. However, since every arrangement discussed above yields the same or similar expressions of the average oxygen diffu-

Fig. 22.4 Combined compartment model of oxygen tensions in brain. The compartments include arterial, capillary, venous, and mitochondrial spaces. The interface between the capillary and mitochondrial compartments is a diffusion barrier, the exact position of which is not known with certainty, although it may be dominated by the capillary endothelium. The numbers refer to normal oxygen tensions (mmHg), calculated from Eq. (22.7) for capillary oxygen tension,

Eq. (22.5) for venous oxygen tension (z = 1), and Eq. (22.19) for mitochondrial oxygen tension (x = 1). The oxygen tension of the average tissue compartment or capillarymitochondrial diffusion interface is a simple linear average. Note that the term capillary bed is used for the entire portion of the vascular bed that interacts with the tissue. This portion may include elements of arterial microvessels (Duling and Berne 1970).

22.4 Flow-Metabolism Coupling of Oxygen

sibility, it appears generally and for most practical purposes valid to estimate the lowest oxygen tension in the tissue (which may also be the tension of the uniformly distributed oxygen, of course) by the formula mit cap O P P O2 2

JO2 L

22:20

mit is either the uniform tissue oxygen tension in the linear case, or in which P O2 the minimum tissue oxygen tension in the nonlinear case. The average tissue oxygen tension, in contrast, depends on the actual oxygen distribution but generally must observe the following inequalities: cap P O2

2JO2 br cap < PO2 < PO2 3L

JO2 2L

22:21


The expressions for the blood-brain transfer and metabolism of oxygen must be combined to obtain a general expression of the flow-metabolism coupling of oxygen. In this coupling, the mitochondrial oxygen tension represents the balance between the diffusion of oxygen to the site of metabolism and the reaction of oxygen with cytochrome c oxidase, known to follow the simple MichaelisMenten expression given above. Solution of Eq. (22.9) for the cerebral blood flow yields: 0 " # 1 mit h JO2 LP JO2 O2 A F art @1 P cap 2CO2 L 50

22:22

where F is the blood flow to the tissue (per unit weight or volume), JO2 is the average oxygen metabolism of the tissue, COart2 is the arterial oxygen concentra is the oxygen diffusibility, P cap is the half-saturation oxygen tension of tion, L 50 hemoglobin in capillaries, corrected for the Bohr shift and prior arterial desat is the weighted average of Hill’s coefficient for the capillary uration, and h bed.3) When the two equations involving the uniform or minimum oxygen tenmit O can be eliminated from the relasion are linked (Eqs. (22.10) and (22.22)9, P 2 may compensate for the de3) Adjustment of L gree of hypoxemic desaturation of arterial P app can be conblood, provided the product L 50 sidered a constant. This is the case if the magnitude of L is adjusted to compensate for app by desaturation, by a prothe decline of P 50

cess of recruitment. Equation (4) shows that the com-bined desaturation prior to capillary entry and a commensurate adjustment of the bloodbrain oxygen diffusion capacity together could serve to render the baseline magnitude of the product valid for all degrees of desaturation.

535

536


Fig. 22.5 Flow-metabolism couple predicted by Eqs. (22.22) and (22.23) for human whole brain and visual cortex. Parameters used to generate curves are given in Tables 22.1 and 22.2 for whole human brain and human cerebral cortex. Abscissa: blood flow (ml hg–1 min–1). Ordinate: oxygen consumption (lmol hg–1 min–1). Points indicate normal values listed in Table 22.2 for the two tissues. Change from whole-brain to cortex is associated with slight decline of the oxygen extraction fraction (EO2 declines from 0.39 to 0.38).

tionship. The resulting formula prescribes the rate of blood flow that is associated with given properties of cytochrome c oxidase: 0 JO2 F art @1 2CO2

"

1 # !h cyt JO2 LP 50 A 1 cap Jmax JO2 LP 50

22:23

where all the parameters in principle are variables under homeostatic control. The variables have all been defined above. The equation describes a mechanism of nonlinear flow-metabolism coupling, cast in terms of the nominally indepencyt cap and the nominally Jmax, P50 , and h, dent variables or constants F, COart2 , P50 , L, dependent variable JO2 . If independent variables other than flow remain constant, oxygen consumption must follow flow nonlinearly. As oxygen metabolism is known not to depend rigidly on blood flow, for example in hypocapnia, it is safe to conclude that several independent variables undergo simultaneous


Fig. 22.6 Changes of oxygen consumption and blood flow, relative to respective baselines for human whole-brain, calculated from human wholebrain curve shown in Fig. 22.5. Abscissa: change in blood flow, relative to baseline (%). Ordinate: change in oxygen consumption, relative to baseline (%). Note approximate linearity, the slope of which is close to the reciprocal of magnitude of the average capillary Hill coefficient 1=h, as indicated by Eqs. (22.22) and (22.23).

change. One possible adjustment is the compensation for low oxygen saturation cap for as argued app for P of arterial blood suggested by the substitution P 50 50 4) above. In principle, the relationship between oxygen consumption and blood flow described by the flow-metabolism couple is flow-limited. It is shown in Fig. 22.5 for normal values of the independent variables and constants for human brain. Assuming these parameters to be invariant, the relationship is highly nonlinear and fixed. The changes of oxygen consumption, relative to baseline, in contrast, 4) The complete flow-metabolism equation

shows that the effect of arterial hypoxemia can also be counteracted by maintaining a cyt mit mit constant PO =P50 ratio as PO declines. It is 2 2 of interest that experimental observations conform less well to the equation when it is

correctly modified for the prior arterial desaturation of hemoglobin. This suggests either that reserves of oxygen are available in the tissue for use by the mitochondria, or that “recruitment” to compensate for the prior arterial may reset L desaturation of hemoglobin.

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538


and the accompanying changes of blood flow, also relative to baseline, are linearly linked, as shown in Fig. 22.6, indicating that the assumption of invariant variables and constants other than blood flow and oxygen consumption is valid under these conditions. It is increasingly clear, however, that the independent variables as well as the constants must undergo substantial changes in some circumstances. Independent variables or constants that may undergo simultaneous adjustment include the cytochrome oxidase maximum reaction rate or affinity, or both. There is evidence that they change in the same direction during the activation of cytochrome oxidase in vitro, while there is other evidence that the affinity of cytochrome oxidase is subject to inhibition by nitric oxide, which is also likely to play a major role in flow adjustment (Gjedde et al. 2005).

22.5 Limits to Oxygen Supply

Oxygen consumption is secured by an adequate blood flow and the possible adjustments of independent variables in Eq. (22.23). However, these adjustments eventually are exhausted if the flow declines to a certain threshold, below which the compulsatory flow-limitation of oxygen consumption intervenes. At that threshold, it is no longer possible for the circulation to maintain normal oxygen metabolism of all parts of the tissue. The imbalance may be due to insufficient capillary driving pressure or increased maximum reaction rate, or both, and the effect of the imbalance is different for the different arrangements and regulations of mitochondria and cytochrome oxidase discussed above. However, in all cases, the oxygen tension will decline until no mismatch exists between the metabolism and the delivery, but the needs of tissue will not be satisfied in part or in whole.

22.5.1 Distributed Model of Insufficient Oxygen Delivery

The issue of when the needs of the tissue are not satisfied can be resolved by consideration of the tissue oxygen distribution. Equations (22.11) to (22.15) apply to the situation in which the capillary oxygen supply is not sufficient to supply the entire tissue at the necessary rate. When that happens, the segmental fraction x served with oxygen will not reach unity (x < 1). In the case of nonuniform oxygen distribution, the effect depends on the distribution of metabolism. It is possible that some parts of the tissue exhaust the oxygen supply, leaving other parts without oxygen. The exhaustion of the oxygen supply by a given fraction (x) of the tissue mass or volume can be predicted from Eq. (22.15) by introduction of a limit, x0 , at which the oxygen supply is exhausted (x0 < 1):


PO2 x

x 2 x0

cap PO2

2 ! JO2 x x0 L

22:24

where JO2 is the normal rate of oxygen consumption of the tissue, excluding the part that is not supplied with oxygen (equal to 1 x0 ), and x0 is the fraction consuming oxygen at the normal rate. The oxygen tensions accompanying the depletion of the oxygen supply are given by Eq. (22.24), which yields the value of x (x0 ) at which the tension reaches zero, when the supply does not match the demand. The actual oxygen consumption average of the tissue is then JO0 2 x0JO2 . In this case, Eq. (22.1) is no longer valid as written but must be modified to account for the nonlinear rise of the extraction fraction caused by the exhaustion of the oxygen supply. Equation (22.1) modifies to Z zJO2 x0 zdz EO2 z FCOart2 z

22:25

0

where as above JO2 is the normal oxygen consumption per unit mass or volume of tissue. To solve Eq. (22.25) for x0 (z), it is necessary to establish the capillary oxygen tension as a function of the extraction fraction reached in segment (z) serving the volume fraction x according to Eq. (22.5): cap PO2 z

s 1 1 EO2 z

app P50 zhz

22:26

as well as the fraction of the tissue supplied with oxygen at that tension accordmit O 0 (x0 £ 1): ing to Eq. (22.6) for P 2 s cap LzPO2 z x0 z JO2

22:27

where Lz and JO2 are assumed to be known from the normal cases of adequate oxygen supply. Thus, when Lz and JO2 are known, numerical solution of the three equations (Eqs. 22.25 to 22.27) with the three unknowns cap EO2 z, PO2 z and x0 (z) yields both the capillary oxygen tension profile for a case of insufficient oxygen supply and the resulting oxygen consumption JO0 2 z x0 zJO2 . The solution can be further illustrated by consideration of the flux capacity by differentiation of Eq. (22.24), which yields a description of the oxygen metabolism in each segment of the tissue (x) at each segment of the capillary bed (z): jO2 z; x x0 z

xJO2

cap

LzPO2 z x0 z

xJO2

22:28

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Fig. 22.7 Flux capacitor lines for distributed model of tissue oxygen, predicted from normal values shown in Tables 22.1 and 22.2, and in conditions of reduced blood flow as indicated in the graph. Dependent variable was calculated by means of Eqs. (22.11) and (22.28). Abscissa: tissue segment (x) served by average capillary compartment (z = 0.5). Ordinate: oxygen flux as a function of x. Flux reserve at normal blood flow is consistent with minimum mitochondrial oxygen tension of 8.5 mmHg. At the lowest blood flow rate, only 96% of tissue (x0 = 0.96) can consume oxygen at the normal rate.

which is a line of slope JO2 and ordinate intercept JO0 2 z. The ordinate intercept is the actual oxygen flux from that part of the capillary bed, equal to the product of the capillary oxygen pressure and oxygen diffusibility, relative to the tissue fraction served at that point [x0 (z) £ 1]. The linearly declining flux capacity of Eq. (22.28) is illustrated in Fig. 22.7 for normal human brain as well as for lowered blood flow rates, including a blood flow rate just below the ischemic threshold. For values of z at which x0 < 1, EO2 z fails to rise linearly and the compartmental simplification is then possible only by numerical solution of Eqs. (22.25) to (22.27).


22.5.2 Compartment Model of Insufficient Oxygen Delivery

In the case of uniform oxygen distribution, the reaction rate declines in every mitochondrion until the consumption again matches the supply, as dictated by the flow. The extraction of oxygen at which this happens can be evaluated by O , assuming mit equal to zero and solving for E means of Eq. (22.9) by setting P 2 O2 diffusibility and hemoglobin saturability to remain constant. The threshold and the declining oxygen consumption below the threshold can also be illustrated as a contraction of the tissue fraction served by the capillary compartment. This is shown in Fig. 22.8 as a diminuation of the capillary segmental fraction z served by the capillary compartment, beginning at the blood flow rate at which x declines below unity before the end of the capillary bed has been reached.

Fig. 22.8 Prediction from Eq. (22.1) of apparent ischemic contraction of magnitude of effective capillary bed serving brain tissue, using standard values listed in Tables 22.1 and 22.2. Abscissa: cerebral blood flow (ml hg–1 min–1). Ordinate: capillary bed segments serving entire tissue segment (x0 = 1). Effective capillary bed starts contracting at blood flow rates below 27 ml hg–1 min–1.

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The actual oxygen flux is less than the arterial supply of oxygen, because it depends on the average oxygen tension in the capillary, which in turn depends on the extraction of oxygen. The discrepancy between the arterial delivery of oxygen and the actually available oxygen supply is a result of the observed diffusion-limitation of oxygen delivery to brain tissue (Gjedde et al. 1991; Kassissia Thus, while oxygen consumpet al. 1995), as expressed in the magnitude of L. tion is flow-limited, oxygen delivery is diffusion-limited.

22.6 Experimental Results 22.6.1 Brain Tissue and Mitochondrial Oxygen Tensions

Using values listed in Tables 22.1 and 22.2, the inequality given as Eq. (22.21) makes the prediction that average oxygen tensions in human brain tissue range over 20–30 mmHg, depending on the distribution and properties of oxygen consumption sites. Measurements of oxygen tensions date back many decades for animal brain, but are not particularly well described in human brain. As expected from the treatment leading to inequality (Eq. 22.21), most authors found that values in human brain tissue in vivo range from 100 mmHg close to arterioles to less than 30 mmHg further from the microvessels. However, the majority of values lie in the range of 20–40 mmHg (Zauner et al. 2002), which is completely consistent with the tissue compartmental average of 30 mmHg summarized in Fig. 22.4. Mitochondrial oxygen tension in human brain tissue in vivo can only be inferred indirectly from the oxygen-binding properties of hemoglobin and the oxygen-metabolizing properties of cytochrome c oxidase. These are summarized in Table 22.1. Using these values and the well known ischemic threshold of oxygen extraction of 60% (see below), Gjedde et al. (2005) employed Eq. (22.19) to estimate an average mitochondrial oxygen tension of 8.5 mmHg in human brain.

Table 22.2 Average properties of human whole brain and cerebral cortex. (Modified from Kuwabara et al. 1992), Vafaee et al. 1999), and Gjedde et al. 2005). Variable (unit)

Whole brain

Cerebral cortex

CMRglc (lmol hg–1 min–1) CMRO2 (lmol hg–1 min–1) CBF (ml hg–1 min–1) Oxygen extraction fraction (EO2; ratio) Oxygen diffusibility (L; lmol hg–1 min–1 mmHg–1) Cytochrome oxidase activity (Jmax; lmol hg–1 min–1)

0.25 1.40 0.43 0.39 0.032 1.59

0.30 1.60 0.50 0.38 0.041 1.70

22.6 Experimental Results

22.6.2 Flow-Metabolism Coupling

Equation (22.23) is the quantitative expression of the flow-metabolism couple in compartmental kinetics. The equation considers only the steady-state, although it could possibly be used to predict some transient events, provided the rate constants of the transients were of the appropriate magnitude. The foundations of the equation is shown in Figs. 22.1 to 22.3. Figure 22.1 shows that the oxygen extraction as a function of capillary segment rises linearly. Fundamentally similar models of non-linear flow-metabolism coupling have been formulated by several authors (Weibel 1984; Buxton and Frank 1997; Gjedde 1997; Vafaee and Gjedde 2000; Aubert and Costalat 2002). The inverse relation between the oxygen extraction fraction and the oxygen tension of the capillary bed is central to these revisions of the conventional coupling of flow to

from Eqs. (22.7) Fig. 22.9 Estimation of whole-brain oxygen diffusibility (L) and (22.8), using standard values listed in Tables 22.1 and 22.2. Abscissa: possible magnitudes of oxygen diffusibility (lmol hg–1 min–1 mmHg–1). Ordinate: predicted oxygen extraction fraction threshold as a function of possible oxygen diffusibility. The known ischemic oxygen extraction limit (E0) of 60% is consistent with a magnitude of L of 3.17 lmol hg–1 min–1 mmHg–1.

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Fig. 22.10 Flow-metabolism coupling during stimulation of visual cortex of human brain, measured with positron emission tomography of labeled oxygen uptake. Curve shows segment of cortical flow-metabolism couple illustrated in Fig. 22.5 for cerebral cortex. Abscissa: cortical blood flow (ml hg–1 min–1). Ordinate: oxygen consumption in human visual cortex (lmol hg–1 min–1). (Modified from Vafaee et al. 1999).

metabolism, which is assumed to maintain a normal oxygen extraction fraction. The revisions arise from the insight that an inverse relation is necessary because every increase in the extraction fraction represents a reduction in the partial pressure of oxygen in the capillary compartment. The formulation of the steady-state flow-metabolism couple is consistent with evidence obtained in visual cortex activation (Vafaee et al. 1999; Gjedde and Marrett 2001), motor cortex activation (Kastrup et al. 2002; Vafaee and Gjedde 2004), and hypothermia (Sakoh and Gjedde 2003), as shown in Figs. 22.9 to 22.13. The evidence shown in Fig. 22.10 is consistent with human cortical gray matter having an oxygen diffusibility of 4 lmol kg–1 min–1 mmHg–1, while the evidence shown in Fig. 22.11 is based on the slightly higher whole-brain oxygen diffusibility of approximately 5 lmol kg–1 min–1 mmHg–1 in the smaller pig brain. The evidence also confirms that blood flow rates are down-regulated when the energy demand of neurons falls at low temperatures (Sakoh and Gjedde 2003). The formulation of Eq. (22.23) is also consistent with evidence from functional MRI experiments which yield changes in oxygen consumption relative to baseline oxygen consumption as a function of changes in blood flow relative to

22.6 Experimental Results

Fig. 22.11 Flow-metabolism coupling during hypothermia, calculated for porcine cortical tissue with oxygen diffusibility of 5 lmol hg–1 min–1 mm–1 Hg. Abscissa: oxygen consumption (lmol hg–1 min–1). Ordinate: cortical blood flow (ml hg–1 min–1) and cerebral core temperatures (8C). Note that axes have been inverted from previous graphs). (Modified from Sakoh and Gjedde (2003).

the baseline blood flow. This evidence generally shows that the relative change of the oxygen consumption is a linear function of the relative change of blood flow. Equation (22.23) makes the prediction illustrated in Fig. 22.6, in which the slope of the relative oxygen consumption change is 27% of the relative blood flow change for whole human brain. This is similar to the 30% relation reported by Kastrup et al. (2002), shown in Fig. 22.12, but lower than the 50% relation reported for human visual cortex by Hoge et al. (1999), shown in Fig. 22.13. It is important to keep in mind that a linear relation between changes in oxygen consumption and blood flow with a slope different from unity, relative to the respective baselines, is proof of a nonlinear relation between the absolute values of the respective variables.

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Fig. 22.12 Relative changes of blood flow and oxygen consumption in human motor cortex, measured by functional MRI (Kastrup et al. 2002) and positron emission tomography (Vafaee and Gjedde 2004). Abscissa: change in blood flow, relative to baseline (fractions). Ordinate: change in oxygen consumption in human motor cortex activation by finger motion, relative to baseline (fractions). (Modified from Kastrup et al. 2002 and Vafaee and Gjedde 2004).

22.6.3 Ischemic Limits of Oxygen Diffusibility

Figures 22.2 and 22.3 (and 22.8) show that cerebral blood flow to the human brain limits the oxygen delivery below a threshold of 25 ml kg–1 min–1, corresponding to an oxygen extraction of 60%. Below this rate of blood flow, the oxygen supply is not sufficient to satisfy the demand. For standard values of metabolism and diffusibility, listed in Tables 22.1 and 22.2, the threshold is reached O rises to 60%. The threshold extraction of 60% has been confirmed in when E 2 a number of studies of human brain summarized by Gjedde et al. (2005) as well as in an animal study (Scheufler et al. 2002). The threshold extraction depends on the magnitude of the diffusibility L, which in turn can be inferred from the magnitude of the mitochondrial oxygen mit is mit . Using the well known ischemic limit as an indication that P tension P O2 O2 and the negligible, Eqs. (22.7) and (22.8) give rise to a relationship between L ischemic threshold shown in Fig. 22.9, according to which the magnitude of L is close to 3 lmol hg–1 min–1 mmHg–1 (3.17 lmol hg–1 min–1 mmHg–1 according to Gjedde et al. 2005).

References

Fig. 22.13 Relative changes of blood flow and oxygen consumption during stimulation of human visual cortex, measured by functional MRI. Abscissa: change in blood flow, relative to baseline (%). Ordinate: change in oxygen consumption in human motor cortex activation by photic stimulation of visual cortex, relative to baseline (%). (Modified from Hoge et al. (1999).

References

Aubert A, Costalat R 2002, A model of the coupling between brain electrical activity, metabolism, and hemodynamics: application to the interpretation of functional neuroimaging, NeuroImage 17, 1162–1181. Buxton RB, Frank LR 1997, A model for the coupling between cerebral blood flow and oxygen metabolism during neural stimulation, J Cereb Blood Flow Metab 17, 64–72. Crone C 1963, The permeability of capillaries in various organs as determined by use of the “indicator diffusion” method, Acta Physiol Scand 58, 292–305. Duling BR, Berne RM 1970, Longitudinal gradients in periarteriolar oxygen tension: a possible mechanism for the participation of oxygen in local regulation of blood flow, Circ Res 27, 669–678. Gjedde A, Ohta S, Kuwabara H, Meyer E 1991, Is oxygen diffusion limiting for blood-brain transfer of oxygen? in Brain Work and Mental Activity, Alfred Benzon Symposium 31, eds. Lassen NA, Ingvar DH, Raichle ME, Friberg L, Munksgaard, Copenhagen, pp. 177–184.

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Gjedde A 1997, The relation between brain function and cerebral blood flow and metabolism, in Cerebrovascular Disease, ed. Batjer HH, Lippincott-Raven, Philadelphia, pp. 23–40. Gjedde A, Marrett S 2001, Glycolysis in neurons, not astrocytes, delays oxidative metabolism of human visual cortex during sustained checkerboard stimulation in vivo, J Cereb Blood Flow Metab 21, 1384–1392. Gjedde A 2002, Cerebral blood flow change in arterial hypoxemia is consistent with negligible oxygen tension in brain mitochondria, NeuroImage 17, 1876– 1881. Gjedde A, Marrett S, Vafaee M 2002, Oxidative and nonoxidative metabolism of excited neurons and astrocytes, J Cereb Blood Flow Metab 22, 1–14. Gjedde A, Johannsen P, Cold GE, Ostergaard L 2005, Cerebral metabolic response to low blood flow: possible role of cytochrome oxidase inhibition, J Cereb Blood Flow Metab 25, 1183–1196. Gnaiger E, Steinlechner-Maran R, Mendez G, Eberl T, Margreiter R 1995, Control of mitochondrial and cellular respiration by oxygen, J Bioenerg Biomembr 27, 583–596. Gnaiger E, Lassnig B, Kuznetsov A, Rieger G, Margreiter R 1998, Mitochondrial oxygen affinity, respiratory flux control and excess capacity of cytochrome c oxidase, J Exp Biol 201, 1129–1139. Guilivi C 1998, Functional implications of nitric oxide produced by mitochondria in mitochondrial metabolism, Biochem J 332, 673–679. Hansen AJ 1984, Ion and membrane changes in the brain during anoxia, Behav Brain Res 14, 93–98. Hansen AJ 1985, Effect of anoxia on ion distribution in the brain, Physiol Rev 65, 101–148. Hoge RD, Atkinson J, Gill B, Crelier GR, Marrett S, Pike GB 1999, Linear coupling between cerebral blood flow and oxygen consumption in activated human cortex, Proc Natl Acad Sci USA 96, 9403–9408. Kassissia IG, Goresky CA, Rose CP, Schwab AJ, Simard A, Huet PM, Bach GG 1995, Tracer oxygen distribution is barrier-limited in the cerebral microcirculation, Circ Res 77, 1201–1211. Kastrup A, Krüger G, Neumann-Haefelin T, Glover GH, Moseley ME 2002, Changes of cerebral blood flow, oxygenation, and oxidative metabolism during graded motor activation, NeuroImage 15, 74–82. Krogh A 1919, The number and distribution of capillaries in muscles with calculations of the oxygen pressure head necessary for supplying the tissue, J Physiol 52, 405–415. Kuwabara H, Ohta S, Brust P, Meyer E, Gjedde A 1992, Density of perfused capillaries in living human brain during functional activation, Prog Brain Res 91, 209–215. Sakoh M, Gjedde A 2003, Neuroprotection in hypothermia linked to redistribution of oxygen in brain, Am J Physiol 285, H17–H25.

References

Scheufler KM, Rohrborn HJ, Zentner J 2002, Does tissue oxygen-tension reliably reflect cerebral oxygen delivery and consumption? Anesth Analg 95, 1042– 1048. Vafaee MS, Meyer E, Marrett S, Paus T, Evans AC, Gjedde A 1999, Frequencydependent changes in cerebral metabolic rate of oxygen during activation of human visual cortex, J Cereb Blood Flow Metab 19, 272–277. Vafaee MS, Gjedde A 2000, Model of blood-brain transfer of oxygen explains nonlinear flow-metabolism coupling during stimulation of visual cortex, J Cereb Blood Flow Metab 20, 747–754. Vafaee MS, Gjedde A 2004, Spatially dissociated flow-metabolism coupling in brain activation, NeuroImage 21, 507–515. Wang CY, Bassingthwaighte J 2001, Capillary supply regions, Math Biosci 173, 103–114. Weibel ER 1984, The Pathway For Oxygen, Harvard University Press, Cambridge, Mass. Zauner A, Daugherty WP, Bullock MR, Warner DS 2002, Brain oxygenation and energy metabolism, part I: biological function and pathophysiology, Neurosurgery 51, 289–302.

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23 Functional Brain Imaging Gerald A. Dienel

23.1 Molecular Imaging of Biological Processes in Living Brain 23.1.1 Introduction

Specialized cells in the brain are integrated within a complex, intricate organization of cellular networks and pathways designed to carry out many diverse activities. Modern techniques to visualize human brain have their roots in histochemical and autoradiographic methods used to assess “static structure” (e.g., anatomical localization and relative amounts of cell types, specific proteins, lipids, enzymes, receptors, and biological compounds including metabolites and signaling compounds) and “dynamic functions” (e.g., local rates of blood flow, glucose utilization, and oxygen consumption; DNA, RNA, lipid, amino acid, neurotransmitter, and protein synthesis; and changes in concentrations of levels of metabolites, ions, and signaling molecules) of brain (Fig. 23.1). Imaging of biological processes in vivo is particularly important because the properties and integrative activities of the system of interest can be assessed within the environment of the living brain.

23.1.2 Molecular Imaging

Major goals of neuroscientists who use functional imaging as an experimental tool are to understand behavioral and cognitive functions in terms of cellular activities, interactions of neurons, astrocytes, oligodendroglia, and microglia, and interactions of networks of cells. Broad application of imaging techniques to studies of brain function has been a strong driving force to elucidate the electrophysiological, biochemical, and physiological basis for imaging signals. Blood flow, metabolism, and cellular functions are inseparable aspects of brain activity, Blood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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23 Functional Brain Imaging

Fig. 23.1 Functional imaging techniques obtain specific types of information about the brain and its functions. An integrated approach is necessary to build a framework that facilitates studies spanning the range from global to regional, cellular, subcellular, and molecular analysis.

and these processes are targets of molecular imaging because they underlie and contribute to signals used to construct images of living brain at work (Fig. 23.2). Molecular imaging, as defined by Massoud and Gambhir [1], is “the visual representation, characterization, and quantification of biological processes at the cellular and subcellular levels within intact living organisms.” Molecular imaging techniques interface with many disciplines and use a wide variety of probes and technologies designed to detect and measure specific molecules, processes, and functions in cells ranging from gene expression to cognition (Fig. 23.3).

23.1.3 Influence of Blood-Brain Interface on Functional Imaging

Molecular probes to assay brain activities in vivo fall into two major categories: (1) intrinsic compounds that are endogenous components of the blood or brain, and (2) extrinsic compounds that must be inserted into the brain across the blood-brain interface that serves as a protective system for the brain. Together, the blood-brain barrier and the blood-cerebrospinal fluid (CSF) barrier limit the types, amounts, and time-courses of transfer of material from blood to brain by

23.1 Molecular Imaging of Biological Processes in Living Brain

Fig. 23.2 Interrelationships of cerebral blood flow, metabolism, and brain function. Neural activity requires blood flow and metabolism to provide the fuel for energy-dependent functions. Because local rates of blood flow and metabolism are closely linked to local changes in functional activity they are commonly used as indirect measures of brain function.

various mechanisms. For example, the tight-junction blood-brain barrier system blocks entry hydrophilic substances into brain unless there are specific transporters, thereby excluding neuroactive compounds present in blood, such as glutamate, from entering brain. These transporters control the types and quantities of molecules that enter brain by means of their amounts and substrate affinities (Vmax and Km): saturation of a transporter limits uptake into brain when blood levels rise and changes in the number of transporters can help adapt to chronic changes in blood levels of compounds required by brain. Metabolism at the blood-brain interface can also restrict entry into the brain by converting molecules that diffuse through the lipid barrier into other compounds. For example, ammonia readily diffuses into brain but is rapidly converted to glutamine by astrocytes, presumably in their endfeet-surrounding capillaries. Extrusion mechanisms mediated by P-glycoprotein remove specific compounds that gain access to the brain. Although blood-brain barrier systems eliminate use of certain tracers, properties of the blood-brain interface can be used to advantage to design and use extrinsic probes to measure specific processes. The following sections describe imaging methods, then give examples of biological processes actively being studied, along with some emerging approaches.

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Fig. 23.3 Brain imaging can employ one or more methodological approaches to evaluate biological processes associated with specific functions. Tools depend on the technological approach and biochemical or physiological process of interest.

Emphasis is placed on in vivo assays of functional activities, but some in vitro studies in brain slices are also described, since these extend analysis of in vivo findings and serve as driving forces for new in vivo studies. Interested readers are directed to the reference materials for more comprehensive coverage of these topics.

23.2 Overview of Brain Imaging Methodologies

Various technologies, including autoradiography, optical imaging of signals derived from intrinsic and extrinsic molecules, single photon emission tomography (SPECT), positron emission tomography (PET), X-ray computed tomography (CT), magnetic resonance imaging (MRI), and functional magnetic resonance imaging (fMRI) are used, both alone and increasingly more commonly in combination, to obtain pictorial representations of brain structure and functional activity under normal and abnormal conditions in experimental animal and human subjects. Many physiological, behavioral, and cognitive activities can only be studied in brains of conscious subjects and brain imaging is used to investigate and map brain functions that require developmental and experiential


Fig. 23.4 Spatial resolution and temporal discrimination vary with methodological approaches to study brain function. Optical imaging techniques (voltage-sensitive dye imaging, assay of intrinsic signals, or fluorescent dyes) can detect rapid changes with high resolution and repeated measures can be made over long time intervals, depending on experimental design, conditions, and technology. Functional magnetic resonance imaging [fMRI] can detect rapid (< 1 s) changes in blood oxygenation and repeated measures. Radiochemical assays for blood flow and oxygen utilization assays are carried out over intervals of about 1–2 min, whereas metabolic imaging with

deoxyglucose [DG], fluorodeoxyglucose, and other labeled compounds requires 30–60 min. Most of the signal is acquired within the first 5–10 min after pulse labeling; and the remainder of the experimental interval allows clearance of unmetabolized precursor and a longer time to reduce the impact of estimates of rate constants on calculated rates. Parallel assays to assess electrophysiological activity (patch clamping, electroencephalography [EEG], and magnetoencephalography [MEG]) complement imaging approaches. (Reprinted from [2], with permission from the authors and Nature Publishing Group).

maturation in vivo, as well as evolution of pathophysiological changes. Thus, brain imaging is a very important and widely used technique to simultaneously visualize, localize, and quantify structural and functional properties of the entire brain; imaging is also a valuable diagnostic tool to evaluate debilitating disorders, such as stroke, dementia, and tumors. Imaging technologies measure local concentrations of molecules and their synthesis, metabolic transformation, binding, and change of state with different spatial-temporal scales that vary over a wide range (Fig. 23.4). For example, voltage-sensitive dye imaging has the highest spatial and temporal resolution to measure cortical functional activity in living brain, registering changes within milliseconds at distances of microns [2]. Activity-dependent changes in optical

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properties of brain tissue also have millisecond temporal resolution and autofluorescence arising from changes in the redox state of intrinsic molecules, such as NAD(P)H and flavoproteins, can be registered within seconds and millimeters [3–6]. fMRI detects and localizes rapid shifts in the level of oxygenation of hemoglobin in blood within a few seconds with spatial resolution of a few millimeters that is being improved to the tens of milliseconds and sub-millimeter ranges [7]. Nuclear-based imaging can assess a broad array of metabolic processes, metabolite and electrolyte transport, blood-brain integrity, receptor binding, and gene expression over time-scales of tens of seconds to 5–60 min, with spatial resolution in the order of 50 lm for 14C-based autoradiography to 1–2 mm for PET and SPECT [1]. The different technologies also vary in the depth of tissue that can be imaged and sensitivity to detect changes in the molecular probe.

23.2.1 Computed Tomography

Major techniques to visualize brain structure or anatomy are CT and nuclear MRI. Coaxial tomographic systems measure the attenuation of X-ray beams derived from an external source that pass through a target tissue and are registered on detectors. Tissue composition and water content contribute to mass density that is proportional to the attenuation coefficient, and X-ray transmission data are used for image reconstruction by various techniques. No contrast agents are required and the scan time is relatively short (about 20 s) compared to about 5–60 min for SPECT and PET scan times. CT scans are useful for detection of changes in ventricular volume or shape, regional atrophy, tissue density, and, when combined with PET radiotracer imaging, anatomic and diagnostic information improves interpretation of the imaging studies; use of intravenous contrast agents allows CT blood perfusion and arteriography imaging [8– 10].

23.2.2 Magnetic Resonance Imaging

Magnetic resonance imaging takes advantage of the magnetic properties of atoms with an odd number of protons and/or neutrons (e.g., 1H, 13C, 23Na, 31P) and the ability of atoms to absorb energy at a very specific (resonance) radio frequency (RF) and later emit energy at the same RF after the excitation pulse ceases. The time-constants of the emitted energy are dependent on interactions with tissue, thereby introducing an influence of biological properties of tissue on the emitted signal that can be analyzed and used to construct images [11, 12]. In tissue, hydrogen in water is the most abundant atom and provides the strongest signals that vary in strength with proton density. In their normal envi-


ronment, protons spin about their axis and form magnetic fields that have random orientations. When a strong external magnetic field is imposed these nuclei become aligned with the imposed magnetic field (the longitudinal or z-axis). If an RF pulse with a specific frequency is transmitted to the aligned nuclei in the transverse plane (x-y plane), the nuclei absorb energy if their resonance frequency is the same as the frequency of the RF pulse and they shift the direction of their magnetic moments toward the transverse plane. When the radio frequency pulse is turned off, the nuclei return to the original alignment in the strong external magnetic field and give off energy at the resonance frequency in a time-dependent manner. This decay or relaxation time provides a signal that has two major tissue-dependent components, T1 and T2. T1 reflects the time constant for return to magnetic alignment along the longitudinal or z-axis; and the T1 component of the relaxation process is due to the ability of protons to exchange energy with neighboring protons (spin-lattice relaxation time). The T2 signal reflects loss of signal strength due to loss or dephasing of the spin coherence (spin-spin relaxation time) in the x-y plane after the RF pulse ceases. T2* is the loss of spin phase coherence that arises from inhomogeneities in the magnetic field of the external magnet and in the local environment. Tissue components with a short T1 return to equilibrium faster and give a higher signal, whereas those with long T2 give a stronger coherent signal. In general, high signal intensity arises from high proton density, short T1, and long T2; and a low signal is associated with low proton density, long T1 and short T2. RF pulse sequence conditions have been devised to enhance gray-white matter contrast or detection of abnormalities and images can be constructed so that they mainly reflect T1, T2, or T2* signals (T1-, T2-, or T2*-weighted images); and weighting modalities are especially useful for imaging pathophysiological conditions [11]. Three-dimensional MRI images with high spatial resolution are extremely useful for anatomical images [12], particularly when combined with dynamic imaging technologies that provide functional information [13].

23.2.3 Functional MRI

Blood oxygen level-dependent (BOLD) contrast imaging, or fMRI, has been widely used to visualize neural activity in humans and experimental animals since the methodology was devised by Ogawa and colleagues in the early 1990s [14, 15]. The basis for the method is that oxyhemoglobin in blood is diamagnetic (low magnetic susceptibility), whereas deoxyhemoglobin is paramagnetic and significantly influences the T2*, causing signal attenuation or contrast. Thus, the BOLD signal represents the balance between oxy- and deoxyhemoglobin within a sampling volume, and venous blood with high levels of deoxyhemoglobin appear dark on images. Increased oxidative metabolism relative to oxygen delivery causes a negative BOLD response, whereas a higher supply of oxygen compared to demand causes a positive BOLD response because the level

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Fig. 23.5 fMRI measures BOLD responses to neural activation. The BOLD contrast MRI registers changes in blood oxygenation and can be influenced by the net balance between oxygen supply and demand arising from changes in metabolic activity of brain cells, blood oxygen level, blood volume, blood flow, and other factors. Oxyhemoglobin is diamagnetic and does not contribute to bold contrast, whereas deoxyhemoglobin is paramagnetic and venous blood containing deoxyhemoglobin contributes BOLD contrast and appears dark in the image. An increase in oxygen consumption that exceeds oxygen delivery will cause a negative BOLD response, whereas an increase in blood flow and vascular volume in excess of oxygen utilization will reduce

the amount of deoxyhemoglobin in venous blood and cause a positive BOLD response. Top panels: (a) MRI image showing placement of electrode tip in primary visual cortex of anesthetized monkeys. (b) A localized positive BOLD response (color scale: red > orange > yellow > blue) in striate cortex to rotating checkerboard patterns; activation is measurable around the electrode tip. (c) Hemodynamic (BOLD) response (red line) superimposed on the raw neural signal (black) and the root mean square of the signal (thick yellow line). Neural activity began at the onset of the stimulus (blue line along abscissa) and ceased when the stimulus terminated, whereas the onset of the positive BOLD response lagged by about 2.2 s and peaked


of deoxyhemoglobin in the venous blood is reduced. Functional activity not only influences oxidative metabolism, but also governs regulation of hemodynamic properties, including blood volume, blood flow, and blood oxygen content, all of which can secondarily affect the BOLD signal [14, 15]. Because changes in functional activity are closely linked to hemodynamics and metabolism, fMRI is widely used to map changes in neural activity in human brain, as well in experimental animals (Fig. 23.5). This approach has the advantage that intrinsic signals are measured and repeated studies on the same subject can be carried out within single sessions and in longitudinal studies. The physiological basis of fMRI is not yet fully understood, but emerging evidence indicates that the BOLD signal may or may not show an initial (negative) dip due to rapid oxygen consumption just after onset of activation, whereas the positive BOLD response lags the increase in neural activity by several seconds due to slower hemodynamic responses and it involves a greater tissue volume than the activated cells. The positive signal may reflect neural activity related to input and local processing rather than spiking activity that is thought to be related to output of a functional unit (Fig. 23.5) [16–20]. Exogenous NMR contrast agents extend and complement the use of signals arising from endogenous compounds. For example, a paramagnetic agent, Mn2+, has three major types of applications, assessment of active areas due to uptake of Mn2+ into excitable cells via voltage-gated calcium channels, three-dimensional tracing of anatomical pathways in longitudinal studies due to anterograde transport after direct injections, and whole-brain contrast after a peripheral injection of MnCl2, enabling detailed visualization of cytoarchitecture [21]. Also, contrast derived from superparamagnetic iron oxide (SPIO) can be used to

Fig. 23.5 (continued) later. The relationship between neural activity and the fMRI signal remains to be established, but data analysis indicates that the BOLD response appears to be more strongly correlated with local field potentials that reflect input and local integrative processing of signals in a given area than with neuronal spiking activity that is thought to be related to the output of the area. (Reprinted from [16], with permission from the authors and Nature Publishing Group). Bottom figure: A simple model portraying possible effects of synaptic inhibition on activity-dependent blood flow and BOLD contrast signals. Left panels: Excitatory activity associated with glutamate release activates cellular activity and causes an increase in oxygen consumption, producing deoxyhemoglobin (deoxyHb), thereby leading to a negative

BOLD response because utilization exceeds supply. Activation of voltage-sensitive calcium channels and calcium signaling pathways are postulated to then activate enzymes governing neurovascular regulatory mechanisms leading to increased blood flow and vascular volume, causing a subsequent positive BOLD response (see top panels b and c). Right panels: Activation by glutamate release is associated with GABA-mediated synaptic inhibition that reduces activation of voltagegated calcium channels, blocking the blood flow response. In this case, metabolic demand would still rise without a commensurate rise in blood flow, perhaps resulting in a more prolonged negative BOLD response. (Reprinted from Lauritzen [20], © 2005, with permission from the author and Nature Publishing Group).

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image processes as diverse as apoptosis [22] and macrophage infiltration into damaged tissue [23] (see Section 23.6).

23.2.4 Radionuclide Imaging

Quantitative autoradiography, SPECT, and PET have very important roles in brain imaging studies (Fig. 23.6), and different isotopes are used for these methodological approaches (Table 23.1). Autoradiographic analysis has been used for many decades to visualize and quantify distributions of 3H-, 14C-, and 35 S-labeled compounds in tissues [24, 25], as well as movement of labeled elec-

Fig. 23.6 Radioactive tracers have broad application to molecular imaging studies. Beta (b–) emitters, especially the long halflived isotopes 3H and 14C, are widely used in animal studies because a wide variety of biological compounds can be synthesized and used to study specific pathways and processes by quantitative autoradiographic techniques. This approach has high anatomical resolution (50–100 lm) and can be used as a routine procedure in many laboratories. A disadvantage is that each experimental subject can be used only once. In contrast, external imaging by SPECT or PET permits repeated scans of the same

subject, but the technology is much more expensive. SPECT has lower resolution and a more limited number of labeled tracers, compared to PET. Radiolabeled compounds can be used to measure transport, metabolism, and binding of many compounds with different functions and in various metabolic and signaling pathways and neurotransmitter systems. See Table 23.1 for examples of isotopes, half-lives, and processes assayed. S*, radiolabeled substrate; E, enzyme; ES*, enzyme-substrate complex; P*, labeled product; L*, labeled ligand; R, receptor. For more detailed discussion, see [1, 26–28].

23.2 Overview of Brain Imaging Methodologies Table 23.1 Some examples of radiolabeled tracers and processes assayed with imaging techniques. Values for half lives [117] are rounded off. Nuclide

Half-life

Representative compounds Biological processes

PET 82 Rb

1 min

Rb+

15

2 min

13

10 min 20 min

H2O; O2; CO Ammonia Glucose; methionine, thymidine palmitate, acetate; nicotine; SCH 23390, raclopride, WIN 35428; Carfentanil, Ro 15-1788

O

N C

11

68

68 min

18

110 min

Ga F

SPECT 133 I 67 Ga 133 Xe 99m Tc

13 h 78 h 5 days 6h

Autoradiography 86 19 days; Rb; 54 312 days; Mn; 22 2.6 years; Na; 36 3 ´ 105 years Cl 35 S 87 days 45 Ca 165 days 32 P; 14 days; 33 P 25 days 131 I; 8 days; 125 I 60 days 3

H

14

C

12 years

Blood-brain barrier integrity, cardiac blood flow, integrity Blood flow; oxidative metabolism; blood volume Nitrogen and amino acid metabolism Glucose utilization; protein synthesis, DNA synthesis; fatty acid metabolism, oxidative metabolism; nicotinic receptors; dopamine D-1 and D-2 receptors, dopamine transporter; opiate receptor, benzodiazepine receptor Gene activation, protein binding

Ga-chelates, antisense oligonucleotides F–; Bone scan; fluorodeoxyglucose (FDG); glucose utilization; fluoroDOPA, fluorouracil neurotransmitter turnover; nucleic acid turnover Radiopharmaceuticals Ga-chelate Xe Radiopharmaceuticals

Blood flow, receptor binding Tumor detection, localization Regional blood flow Tumors, blood flow

Rb+; Mn++; Na+; Cl– Methionine Ca++ PO–4; ATP Iodoantipyrine

Electrolyte transport and homeostasis

Carbohydrates, lipids, nucleic acids, carboxylic acids, etc. 5 730 years Carbohydrates, lipids, nucleic acids, carboxylic acids, etc.

Protein synthesis Calcium homeostasis Phosphorylation reactions Blood flow; iodination reactions to label proteins, thyroxin, and other compounds Metabolism, signaling, cell division, pharmacology, transport, enzyme mechanisms Blood flow, metabolism, transport, signaling, cell division, receptor pharmacology, enzymes, protein, lipid, DNA, RNA synthesis

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trolytes within the body. In brief, the weak beta particles (electrons) emitted during the decay process can be detected by autoradiography using X-ray film, emulsion, or phosphoimager techniques. Autoradiography provides a relatively cheap and easy-to-use procedure for the routine laboratory setting, but it has the disadvantage that external detection of the low-energy beta particles is not possible and tissue from each experimental subject must be processed for analysis; only one time or treatment sample can be obtained for each animal. The external detection of high-energy c-rays emitted from within the subject is exploited by SPECT and PET. These modalities require expensive equipment and a highly-trained technical staff to carry out the studies but have the advantage that repeated, noninvasive studies can be carried out in the same subject. Single photon c-emitting isotopes (Table 23.1) can be incorporated into various molecules and detected and quantified by c-cameras that rotate around the subject so that tomographic images can be produced. PET has an advantage over SPECT in that many more positron-emitting isotopes that have different, short half-lives (Table 23.1) can be used by radiochemists to synthesize a large variety of labeled compounds of biological importance and interest. Also, the annihilation reaction between a positron and electron produces two photons with an energy of 511 keV emitted at about 180 8 (Fig. 23.6); and these signals are nearly simultaneously detected, then processed to calculate the origin and magnitude of the signal source. The PET detection system, although complex and expensive, can be used for all compounds of interest due to the same 511 keV photons derived from each positron emitter. Extraordinary advances have been made with PET during the past several decades and recent comprehensive reviews and monographs describe progress in this field in detail; many types of radiolabeled tracers are available for evaluating blood components, blood-brain barrier transport, blood-CSF transport, and brain structure and functions [1, 26– 28].

23.2.5 Optical Imaging

Neural activity causes electrochemical, biochemical, and physiological changes in tissue that are detectable by altered absorption, light scattering, fluorescence, and other optical parameters (Fig. 23.7), and optical signals can be generated by activity-dependent electrolyte- and metabolism-driven changes in properties of intrinsic or extrinsic molecules [3–6]. For example, light scattering properties of tissue are altered by changes in ion and water flux across cellular membranes. Oxidative metabolism causes deoxygenation of hemoglobin and changes in its absorption or reflection of near-infrared light; oxidation of NADH and NADPH produces nonfluorescence compounds, whereas oxidation of FADH2 yields a fluorescent product [29]. Near-infrared spectroscopy (NIRS), with a spectral window range of 700– 1000 nm, was first used for noninvasive in vivo optical imaging through the in-


Fig. 23.7 Assays of physiological changes related to brain activity by optical methods. Changes in absorption, fluorescence, and light scattering are the major optical parameters monitored via intrinsic (derived from endogenous compounds) or extrinsic (derived from exogenous molecules) reporter molecules. These optical signals provide information about blood flow, volume, oxygenation, energy status of brain cells, oxidation-reduction status of specific molecules, membrane voltage changes, and other physiological parameters. (Reprinted from [3], with permission from Elsevier).

tact skull by Jöbsis [30]. This approach has less anatomical resolution but good temporal resolution and specificity of the parameters measured. Light absorption is mainly by oxy- and deoxyhemoglobin, water, and lipids, and scattering is due mainly to differences in refractive indices of cellular components [31]. NIRS imaging is sensitive to hemodynamic changes and provides information related to functional activation by noninvasive methods [32]. Real-time imaging of a cerebral thrombosis and vascular leakage after blood-brain barrier damage is achieved using NIR-fluorescent probes [33]. Extrinsic or externally-applied molecules, such as fluorescent dyes, are used to report specific properties of cells related to function-dependent shifts in the free concentrations of major electrolytes and in metabolism (e.g., membrane voltage, calcium level, or pH changes). The uniform loading of exogenous dyes into living brain is difficult because these are lipid-soluble compounds that are not readily delivered by blood; direct application to the brain surface may not result

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in homogeneous distribution of a dye. Also, the magnitude of many fluorescent signals is small, and photobleaching can limit the usefulness of a dye. Nevertheless, voltage-sensitive and calcium-binding dyes are very useful for optical imaging of fast neural events (milliseconds) [2, 34], whereas a pH-responsive dye, neutral red, reports slower changes (seconds) [35]. A recent advance in optical imaging of endogenous fluorofluors is the construction of genetically encoded reporter protein molecules, thereby circumventing the problem of placing the extrinsic compound in the region of interest. Fluorescent protein-based markers expressed in specific cell types are used to monitor cell movements in brain slices [36], neural development [37], and new voltage-sensitive fluorescent fusion proteins are used to assay changes in membrane potential [38]. Fluorescent proteins are mainly used in cultured cells and brain slices with wide-field, confocal, and two-photon microscopy, but future applications will undoubtedly include in vivo studies.

23.2.6 Thermal and Optico-Acoustic Imaging

Infrared imaging of temperature gradients in brain is an emerging technology that is currently being used to detect temperature differences in brain and between brain and tumor. Tumor-tissue temperature differences can arise from the combined effect of hypoperfusion of tissue surrounding a tumor and increased vascularity of tumors [39]. A high resolution infrared camera detects thermal energy that is emitted from a source in proportion to temperature and the resulting electrical signal is displayed as a thermal image on a video monitor. The sensitivity is on the order of 0.02 8C, with a spatial resolution in the 100–400 lm range and scan intervals as low as 10 ms. The brain surface temperature is heterogeneous and the differences between arteries and veins (1.5– 2.0 8C), between veins and parenchyma (0.1–1.0 8C), and between vascular occlusion and reperfusion are readily detectable [39]. Conceivably, this technology might eventually be useful to detect thermal changes arising from functional activation or depression and hemodynamic changes. Another developing technology is ultrasound-mediated imaging that has applications for tumor imaging, but might be useful for functional imaging in the future. Optico-acoustic and photo-acoustic tomography take advantage of the resolution advantage of ultrasound and contrast advantage of optical imaging; biological tissues have poor ultrasound contrast but low attenuation, whereas near-infrared light has better contrast but high scattering and less depth penetration. In brief, the optico-acoustic technology employs very short pulses from a laser light source or radiofrequency source to irradiate tissue, whereas photoacoustic technology employs ultrasonic modulation of coherent laser light in tissue [40]. With optico-acoustic tomography, absorption of the energy from nearinfrared light causes small, focal heating in tissue and this perturbation causes acoustic waves that propagate through the tissue. The acoustic waves are de-

23.3 Imaging Biological Processes in Living Brain: Watching and Measuring Brain Work

tected by ultrasonic transducers and used to reconstruct the optical absorption pattern. Recent studies illustrate the use of endogenous optical contrast (mainly hemoglobin) and exogenous contrast agents, either a dye or gold nanoparticles; and the resultant images can have sub-millimeter resolution at tissue depths as much as 5–6 cm, with the ability to map blood vessels, internal structures, and antibody-bound nanoparticles targeted to tumor cells in vitro [41–43].

23.2.7 Summary

Technologies for imaging brain in vivo (Figs. 23.4 to 23.7) vary widely in their use of intrinsic and extrinsic signaling molecules, their temporal and spatial resolution, the biological parameters that they measure, and the use of conscious or anesthetized subjects. Some approaches are particularly suited for extremely fast events, others for slower processes. In vitro systems and some in vivo systems have key roles for development of more complex and expensive technologies. Some approaches have specific molecular targets that can be measured directly, whereas others assess neural events indirectly, by means of changes in hemodynamics or metabolism. No single methodology or molecular probe can provide a complete picture of the events taking place in brain; and combinations of technologies and imaging probes improve the analysis of functional changes within identifiable anatomical structures over time.

23.3 Imaging Biological Processes in Living Brain: Watching and Measuring Brain Work 23.3.1 Functional Activity, Brain Work, and Metabolic Imaging

In the broadest sense, functional activity involves all brain processes related to information transfer within cells, from cell to cell, and among networks of cells. The term “brain activation” generally refers to an increase in information trafficking and energy demand over and above that measured in a baseline or “resting” state, with implicit recognition that: (1) there is a considerable amount of conscious and unconscious activity taking place in the absence of the specific stimulatory condition or task being studied, and (2) the specific resting state can vary markedly with experimental condition. For example, in certain types of studies, anesthesia is required to prevent movement artifacts during the recording session, and the resting state in the anesthetized subject is clearly not the same as that in the conscious subject. Brain work is mainly related to ionic homeostasis in excitable cells and it has been estimated that 50–60%, perhaps even more, of the energy expended by the brain is used to maintain and restore cellular ion gradients [44–46]. Electrical

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Fig. 23.8 Functional metabolic imaging is based on energy demand and supply. ATP is consumed during functional activity, and glucose and oxygen are the major and obligatory fuels for brain. Oxidative metabolism of glucose yields about 18 times more ATP per mole of glucose than the glycolytic pathway. Thus, small changes in the rates in glucose oxidation or oxygen

consumption, which would be very difficult to measure, can produce as much ATP as much larger changes in glycolysis and lactate production. Endogenous fuel sources, such as glycogen, amino acids, and maybe lactate, can also contribute to the energetics of brain activation; other exogenous fuels, such as blood-borne lactate and acetate, provide some energy.

and molecular events that transmit information via different types of signals from point to point within specific pathways and networks in anatomically defined brain regions consume ATP and produce ADP, thereby requiring the utilization of glucose and oxygen, particularly in the neuropil which is enriched in synaptic endings. ATP is the currency for cellular work (Fig. 23.8) and the turnover of ATP is a critical interface between blood flow, glucose metabolism, neurotransmission, intracellular signaling, and higher brain function (Fig. 23.2). Energy-consuming activities are often broadly characterized in terms of “housekeeping chores” (e.g., maintenance of resting membrane potentials, macromolecule turnover, membrane and vesicle trafficking, and axonal transport) and “stimulus-dependent information processing events” (e.g., ion channel activation and ion pumping, neurotransmitter biosynthesis, release, and re-uptake, and intracellular signaling). Thus, the local functional activation of brain cells and changes in local cerebral blood flow (CBF) and cerebral metabolic rates for oxygen (CMRO2) and glucose (CMRglc) are generally, but not always, closely correlated. Tracers for metabolism and blood flow are, therefore, widely used as surrogates to map the functional activities of cells in the brain in vivo.


Radionuclide-based metabolic mapping is the first and most well established approach that takes advantage of dependence of ATP-dependent activities of brain cells that alter glycolytic and oxidative metabolism of glucose (Fig. 23.8). Increased signaling creates a higher energy demand, mainly in neurons and astrocytes, and stimulates the rate of glucose metabolism in the specific anatomical pathways involved in a task. This, in turn, governs the rate of blood flow to deliver glucose and oxygen to the working cells and causes shifts in the redox state of specific molecules and the relative amount of oxy- and deoxyhemoglobin in the vasculature of activated tissue, giving rise to changes in fMRI and optical signals. Because the magnitude of functional activity or brain work regulates amount of glucose consumed, functional activity, metabolism, and blood flow are sometimes said to be “coupled”. Thus, the rates of fuel delivery and consumption rise when brain signaling increases, whereas they fall when brain function is depressed. Abnormal metabolic rates are often diagnostic of various types of brain disorders, but the usual relationships can change with activation or other conditions. A “metabolic brain image” is a pictorial representation of the activities of all regions of living brain obtained by using a radioactive compound to assay a process of interest. A small amount of radiolabeled tracer is injected into a subject, and with time, the tracer accumulates in tissue in proportion to the rate of blood flow or metabolism. Local tracer concentrations are then measured and used to make an image called a “metabolic map” which reflects the functional activities of the cells in each region of the brain. Thus, functional metabolic mapping can identify and quantify normal and abnormal behavior of cells in specific regions of the living brain in health and disease. Quantitative determination of metabolic rates takes into account the rates of delivery, clearance, and metabolism of the precursor and kinetic differences between a tracer and a natural compound.

23.3.2 Quantitative Measurement of Regional Blood Flow and Metabolism in Living Brain 23.3.2.1 Assays at the Blood-Brain Interface: Global Methods The blood-brain barrier and its transporter systems are major determinants for the delivery of fuel and essential nutrients to brain; many substrates that are readily metabolized by brain slices cannot traverse the blood-brain barrier in sufficient quantities to satisfy the brain’s metabolic demands. Glucose is the predominant, obligatory fuel for adult brain, and without a sustained and adequate supply of glucose and oxygen, brain function quickly fails, causing loss of consciousness, and ultimately, cell death. However, during development or under special circumstances, alternative fuel is consumed by brain. The level of the endothelial monocarboxylic acid transporter (MCT) is high in suckling mammals, and the developing brain metabolizes ketone bodies present in milk. After weaning, the MCT level falls markedly and significant quantities of ketone bodies are utilized by adult brain only during starvation [47, 48]. Essential

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amino acids and some organic acids in blood are also taken up into brain and metabolized, but overall, these are minor energy substrates [47]. Glucose was identified as the major fuel for brain by global methods, i.e., determination of metabolite levels in arterial and venous blood [47]. Quantitative determinations of rates of cerebral blood flow with nitrous oxide [49] permitted the calculation of rates of oxygen and glucose consumption by brain as a whole according to the Fick principle, i.e., CMR = CBF(A-V), where CMR (lmol min–1 g–1 tissue) denotes cerebral metabolic rate for the substance of interest, CBF is cerebral blood flow (ml min–1 g–1), and A and V are the arterial and cerebral venous substrate concentrations (lmol ml–1), respectively. Studies in many laboratories established the overall stoichiometry of utilization of oxygen and glucose, the ratio CMRO2/CMRglc, in normal adult brain of about 5.5 [47]. Because this value is close to the theoretical maximum of 6 for complete oxidation of glucose, most of the glucose entering brain is oxidized. Thus, in adult brain as a whole, only a small percentage of the carbon derived from glucose is used for biosynthetic reactions or is lost from brain, but functional imaging studies have revealed an apparent predominance of glycolytic compared to oxidative metabolism during activation (see Section 23.4.4). The brain is a very heterogeneous tissue and specific functions are localized to small nuclei and neuronal networks. Analytical methods that rely on gross tissue dissection for metabolite analysis therefore lose vital information and can obtain misleading results due to averaging of results obtained in adjacent structures that have unrelated functions or different metabolic rates. Highly sensitive, precise microassays developed for the assay of enzymes and metabolites in individual dissected cells in Oliver Lowry’s laboratory [50] overcame this problem, but the labor-intensive analyses limited the number of samples from each brain that could be analyzed. Development of quantitative autoradiographic methods provided sufficient spatial resolution (about 50–100 lm) and permitted the simultaneous determination of local rates of blood flow and glucose metabolism in all brain structures. This approach not only established a close linkage between functional activity (ATP demand), blood flow, and glucose utilization, but also served as the basis for broad application of quantitative autoradiography as a major tool in neuroscience. Because the basic principles and physiological, pharmacological, anatomical, and pathological applications of the autoradiographic and PET methodology have been reviewed in detail by Louis Sokoloff [51, 52] and others [27, 28], only selected examples will be presented below. The autobiographical perspectives of Seymour Kety [53] and Louis Sokoloff [54], both of whom made seminal contributions to the foundation of the functional brain imaging field, are quite interesting.

23.3.2.2 Highly Diffusible Tracers to Measure CBF The first fully quantitative autoradiographic procedure to measure activity-dependent changes in regional blood flow was developed by Kety and colleagues in 1955 [55]. Tissue levels of [131I]trifluoroiodomethane, an inert radioactive gas, were assayed by autoradiographic analysis of frozen tissue and comparison of


optical densities of regions of interest to those of calibrated labeled standards for determination of tracer concentrations per gram tissue; these values yielded local blood flow rates after dividing by the arterial blood time-activity integral and correcting for the tissue-to-blood partition coefficient for the tracer [55, 56]. Sokoloff and colleagues later developed the use of a less volatile tracer, antipyrine [57] and iodoantipyrine [58]. In the early 1960s, Ingvar and Lassen devised an external scanning method using 85Kr, so CBF could be measured in humans [59, 60]. Blood flow assays are short (< 1 min) and have useful applications in studies to identify brain structures that respond to brief stimuli.

23.3.2.3 Metabolizable Glucose Analogs to Measure Hexokinase Activity and CMRglc 2-Deoxy-d-glucose (DG), a glucose analog lacking the hydroxyl group at carbon 2, was first synthesized in 1920 by Emil Fischer [61], who established some of its physical and chemical properties, such as the extreme acid lability of its glycosidic linkage. In 1954, Sols and Crane [62] assessed the substrate specificity of brain hexokinase and noted that DG isolates the hexokinase reaction, because DG is converted to DG-6-P but not to fructose-6-P. In the late 1950s, Tower used loading doses of unlabeled DG as a competitive inhibitor in assays of glucose utilization and showed the accumulation of its phosphorylated derivative in tissue. In his historical perspective, Tower [63] noted the irony that many researchers who first used DG “failed to see any practical applicability of the effects of this most interesting compound on cerebral metabolism.” [14C]Deoxyglucose was elegantly developed by Sokoloff and colleagues as a tracer to determine local rates of glucose utilization in rat brain [51]. Sokoloff recognized that, under steady-state conditions, the rate of each reaction in a multistep pathway is equivalent, so an assay of the rate of any single step yields the flux through the entire pathway. He developed a theoretical model to describe these processes, analyzed the model mathematically to derive an operational equation for calculating the glucose utilization rate from the rate of DG phosphorylation by hexokinase, and developed the practical procedures to carry out the method using quantitative autoradiography [51]. Within 2 years, [18F]fluorodeoxyglucose (FDG), a positron-emitting analog, was in use for PET studies in primates and man [64–66]. Thus, a number of essential but unrelated studies over a long time span contributed to methodology development. The concept of the DG assay procedure is illustrated in the cartoon in Fig. 23.9. A radiolabeled glucose analog (DG or FDG) is injected intravenously, timed samples of arterial blood are drawn, the quantities of product formed in brain regions of interest are measured, and these values are divided by the integrated specific activity of the precursor pool in brain that is calculated from that measured in blood. Finally, this value for the rate of phosphorylation of the tracer is converted to glucose utilization by correcting for the kinetic differences between glucose and DG or FDG for transport and phosphorylation: approximately two molecules of glucose are phosphorylated to every one of DG. Note

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Fig. 23.9 Metabolic trapping of labeled compounds to measure reaction rates. Tracer levels of radiolabeled molecules that are analogs of endogenous compounds are used to quantitatively measure rates of specific reactions in the midst of all the other biochemical reactions taking place in living brain. 2-Deoxy-d-glucose (DG) is a glucose analog that competes with glucose for: (1) bi-directional transport across the blood-brain barrier and brain cell membranes by glucose transporters and (2) phosphorylation by hexokinase. DG-6phosphate (DG-6-P) is trapped within the cells that metabolize DG for a reasonable duration (about 30–60 min), and in rat brain, about two molecules of glucose are phosphorylated for each molecule of DG [51]. In contrast, products of metabolism of

the endogenous substrate, glucose, are diffusible and rapidly lost from activated tissue, causing large underestimates of functional activation. The design and synthesis of many labeled tracers facilitates the investigation of specific neurotransmitter systems in brain of experimental animals and humans. Targets of interest for molecular imaging tracers, indicated by circles, are: (1) blood (flow, volume, and components, including oxygen levels), (2) astrocytes (metabolism, receptors, or transporters), (3) presynaptic neuronal functions (neurotransmitter biosynthesis and levels, transmitter vesicles, transporters, receptors), and (4) postsynaptic neuronal functions (enzymes, receptors). For more details, see [1, 26–28, 94, 95, 98, 114, 115].

Fig. 23.10 Imaging sensory activation and tumor localization. Left and middle panels, top row: unilateral photic stimulation (15 Hz on/off flash) stimulates the retina and increases neuronal signaling and astrocyte activity in the dorsal superior colliculus of the rat. The local rate of glucose utilization (CMRglc) increases,

reflected by greater trapping of [14C]deoxyglucose-6-phosphate in the activated cells (arrows) but not in the contralateral tissue that was not stimulated. CMRglc measurements reflect the composite activity of all brain cells in the region of interest, whereas acetate metabolism reports oxidative activity in astrocytes due. Astrocyte activity also


Fig. 23.10 (continued) increases during photic stimulation, and is detected by increased metabolic trapping of labeled products, mainly glutamate and glutamine. (Courtesy of N. F. Cruz and G. A. Dienel). Left and middle panels, bottom row: although tumors are highly glycolytic, acetate is rapidly incorporated into TCA cycle-derived amino acids; and tumors can readily be detected with a 5–min labeling period with [2-14C]acetate at 2 weeks after implantation of C-6 glioma cells into rat brain. Because labeling of normal brain tissue by acetate is more uniform than that obtained with [14C]DG, the C-6 glioma tumor-to-tissue labeling ratio was higher for acetate compared to DG. (Courtesy of N. F. Cruz and G. A. Dienel; also see [77]). The color scales represent local tissue 14C concentrations that are proportional to metabolic rates. Note that the four autoradiographs have different color scales (not shown), and 14C concentrations are ranked from highest to lowest in the

following order: red, yellow, green, purple, blue. Right panels: acoustic stimulation increases CMRglc (assayed with FDG) and intracellular uptake of thallium (Tl), a marker for K+ uptake, in gerbil auditory cortex. The top panel shows three tonotopic activation columns in auditory cortex (asterisks show the primary field AI, and arrows show the ventroposterior [VP] field ) in response to a combined 1-kHz and 8-kHz acoustic stimulus. Tl+ (left) and [14C]FDG uptake (right) were measured in the same animal. The lower panel is a pseudo-color image showing thallium uptake in cortical layers of an 8-kHz tonotopic column at higher magnification (scale bar is 50 lm). Arrows in layer II delineate the borders of increased Tl+ uptake and arrows in layer Vb point from a central cluster of pyramidal neuron to satellite clusters. Tl+ uptake is detectable in different cell types (pyramidal neurons, interneurons, putative astrocytes, and dendrites. (Reprinted from [78], with permission from Elsevier).

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that the product trapping of the labeled DG-6-P or FDG-6-P for the duration of the experimental period (30–60 min) is essential for the success of this method. To summarize, development of the first quantitative procedure to measure local metabolic rates simultaneously in all regions of living brain required a comprehensive knowledge of biochemistry, physiology, mathematics, and radiochemistry. Neuroanatomic correlates of processing of physiological information are readily detected and quantified by the DG method, the effects of pharmacological intervention are easily visualized and determined in all brain structures, and glycolytic tumors can be localized. The [14C]DG autoradiographs in Fig. 23.10 illustrate: (1) the regional heterogeneity of glucose metabolism in brain, (2) specific increases in CMRglc in the dorsal superior colliculus in response to unilateral photic stimulus, and (3) localization of a brain tumor by DG. Note the heterogeneous optical densities of the autoradiographs that reflect regional differences in metabolism: the higher the color-coded optical density, the greater the accumulation of product. In normal rat brain, CMRglc ranges from a low of about 0.3–0.4 lmol–1 g–1 min–1 in white matter to about 0.5– 1.8 lmol–1 g–1 min–1 in various gray matter structures, with the highest values in the auditory structures [51]. In primates, values for CMRglc are about half those in the rat but the rank order is similar [52]. In spinal dorsal root ganglia, electrical stimulation increases CMRglc in the synaptic areas, but not in nerve cell bodies, in proportion to the stimulation frequency; and in the pituitary gland, the magnitude of CMRglc was shown to be related to sodium pump activity [52].

23.3.2.4 Non-Metabolizable Analogs to Assay Transport and Tissue Concentration 3-O-methyl-d-glucose is a non-metabolizable analog that is a substrate for glucose transporters. Radiolabeled methylglucose competes with unlabeled glucose for influx into and efflux from brain; and regional glucose transporter activity and glucose concentrations can be quantitatively measured using this tracer. Methylglucose uptake measures transport without the complication of metabolic trapping of the tracer and steady-state levels of methylglucose concentration are quantitatively related to tissue glucose levels. Methylglucose autoradiographs show that glucose concentrations are relatively uniform throughout brain, indicating that glucose supply matches demand (see [67] and cited references therein). The maximal capacity for glucose transport (Tmax) into brain from blood is about three times greater than the maximal CMRglc (Vmax) [68], so supply capacity greatly exceeds demand capacity. Compensatory mechanisms that increase blood flow contribute to the ability of delivery to match and exceed utilization, even with large increases in CMRglc.

23.3.2.5 Cellular Basis of Glucose Utilization A key issue is assessing the contributions of neurons, astrocytes, and other cell types to overall CMRglc during rest, activation, and disease states. Fully quantitative assays with cellular resolution are not possible due to diffusion and loss of


label during tissue processing and staining to identify cell types. [3H]DG studies in the auditory system show no exclusive labeling of one cell type, with high grain density over neurons, fibers, and glia [69]. High-resolution [14C]DG autoradiographic and fluorescence assays with the glucose analog NBDG show similar labeling of neurons and astrocytes under resting conditions [70, 71].

23.3.2.6 Acetate is an “Astrocyte Reporter Molecule” Astrocytes are a major cell type in brain that have essential roles to support and regulate neuronal activity; and development of methods to measure their functional activity in vivo are important. Early studies that evaluated patterns of labeling of glutamate and glutamine by different precursors led to the identification of two major TCA cycles in brain, described as “large” and “small” glutamate pools; acetate labeled the small pool [72, 73]. Acetate and other short-chain carboxylic acids are present in blood and cross the blood-brain barrier via a monocarboxylic acid transporter (MCT) and diffusion. Preferential transport of acetate into astrocytes compared to neurons by the MCT is the basis for celltype specificity of acetate metabolism [74] that has been confirmed by NMR studies [75]. Activation of astrocytes in the dorsal superior colliculus in vivo by photic stimulation of retinal neurons is illustrated in Fig. 23.10. During acoustic stimulation, the calculated rate of oxidative metabolism of acetate in rat brain is similar to estimates of oxidative metabolism of glucose by astrocytes [76], suggesting that acetate is an important minor fuel and it provides biologically significant amounts of energy for working astrocytes. Labeled acetate is also an emerging tool for imaging brain tumors [77].

23.3.2.7 Summary Quantitative autoradiographic assays of cerebral blood flow and metabolism in experimental animals were a major advance in the brain imaging field because they changed data acquisition from mainly “static” information provided by anatomic and histochemical assays to “dynamic” information, consisting of rates of processes that could be simultaneously measured in all regions of conscious subjects under different experimental conditions. A brief history of these methodological approaches was presented to illustrate the complexity of the obstacles facing the initial technological developments that, once surmounted, could be quickly adapted for use in humans. CBF, CMRglc, and CMRO2 are useful measures of overall functioning of brain, but blood flow is not a direct measure of metabolism or of function, and flow and metabolism do not necessarily change proportionately (see Section 23.4.4). Also, the measurement of hexokinase activity does not provide information about the downstream metabolism of glucose, including oxidative metabolism, lactate formation, biosynthetic reactions. Flowmetabolism-function studies paved the way for the development of many sophisticated brain imaging methods and studies, some of which are described in the following sections.

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23.4 Molecular Probes Are Used for a Broad Spectrum of Imaging Assays in Living Brain 23.4.1 Potassium Uptake and CMRglc During Functional Activation

Cellular activation is linked to changes in ion fluxes and increases in K+ uptake during functional activation can be visualized with thallium (Tl+), a K+ analog. Thallium uptake into brain after intraperitoneal injection of thallium acetate is detected histochemically by its precipitation as the sulfide salt during perfusionfixation at the end of the experimental interval [78]. The acoustic stimulation of gerbil with alternating 1 kHz and 8 kHz tones activates specific high and low frequency topographic columns in auditory cortex that are similar in location when visualized with [14C]FDG and Tl2S in the same animal; and at higher magnification, the cellular localization of thallium in neurons of different morphological types and in other cells in cortical layers is readily detected (Fig. 23.10).

23.4.2 Multimodal Assays in Serial Sections of Brain

Brain activation and injury alter many processes that vary in time after the initial event that triggers the biological response, and it is, therefore, essential to be able to quantitatively measure many characteristics of the tissue, as illustrated in Fig. 23.11. One of the advantages of analysis of thin (20 lm) serial coronal sections of brain is that different assays can be carried out in parallel, as long as the tissue preparative method preserves the compounds of interest. Radiolabeling methods are readily combined with fluorescence assays to evaluate the consequences of cortical freezing injury [79] and visualize regional differences in the temporal profiles of immediate and delayed responses (Fig. 23.11). For metabolic studies, the brains must be carefully frozen in situ to preserve labile metabolites that would otherwise be rapidly consumed during the immediate postmortem interval. Alternatively, histochemical and immunohistochemical staining brains can be sampled by routine euthanasia or perfusion-fixation procedures. Local rates of protein synthesis are measured with a radiolabeled precursor amino acid, and if the label on the precursor is not lost via metabolism during the experimental interval, the tissue sections must be washed with acid to remove the unincorporated tracer prior to analysis. Local pH is assayed by fluorescence imaging, whereas ATP, glucose, and lactate levels in freeze-dried sections are assayed by bioluminescence assays after coating the tissue sections with a thin layer of appropriate enzymes and cofactors and exposure to photographic film. Gene expression is assayed by autoradiography after hybridization with 35S-labeled probes for specific mRNAs. Note the severe effects of traumatic brain injury at the site of the lesion, with marked changes in the penumbra re-

23.4 Molecular Probes Are Used for a Broad Spectrum of Imaging Assays in Living Brain

Fig. 23.11 Multimodal assessment of cellular responses after injury. Autoradiographs of coronal sections of rat brain after cortical cold-lesion injury at the level of the striatum (top set) and hippocampus (bottom set) in control (C) and at 1, 3, and 7 days after the lesion. Measured values are: local rates of cerebral protein synthesis (CPS, using [3H]leucine), ATP concentrations (ATPspecific bioluminescence), pH (umbelliferone fluorescence), and c-jun, mkp-1, caspase-3 and GFAP mRNA levels (in situ hybridization with 35S-labeled probes). Note the regionally selective changes in protein synthesis

in the cortex and caudate, local changes in ATP level and pH, and heterogeneous, timedependent responses of gene expression. The cold injury abolished protein synthesis and reduced ATP levels at days 1 and 3 after the cortical lesion (black arrows, left hemisphere; light areas in the autoradiographs), and the surrounding tissue had an increased pH (dark areas in the pH autoradiographs, grey arrows) that extended into more caudal regions (lower panels). Gene expression responses varied considerably. (Reprinted from [79], with permission from Elsevier).

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gion, remote changes throughout the brain, and time-varying effects on different aspects of metabolism and gene expression (Fig. 23.11).

23.4.3 Imaging Human Brain Tumors

One of the most prevalent applications of FDG-PET is localization of tumors in cancer patients. Most tumors are highly glycolytic, so their rates of glucose utilization are generally much greater than surrounding tissue. Tumors, such as melanoma metastases, can be readily located by qualitative assays of [18F]FDG uptake in whole-body scans (Fig. 23.12) that can help guide surgical and treatment approaches, as well as evaluate therapeutic responses [80]. Tumors vary in their extent of vascularization, and the effectiveness of tumor radiation therapy is influenced by tissue oxygen level, so it is important to establish the degree of hypoxia in a tumor for adjustment of the oxygen level in the inspired air mixture to enhance treatment efficacy. Hypoxic tissue is detectable with a PET tracer, [18F]FMISO, that is specifically retained in tissue with low oxygen levels [81]. High labeling of a brain tumor with both FDG and FMISO (Fig. 23.12) is consistent with the presence of hypoxic, glycolytic tissue. One advantage of qualitative FDG tumor localization assays is that the blood time-activity integral does not need to be measured; the patient does not need to be placed in the scanner until about 1 h after the tracer injection, when nearly all FDG has been converted to FDG-6-P. However, this waiting time can lead to unanticipated results, and perhaps, misinterpretation of results unless care is taken. For example, in a case study report [82], a patient who was being assessed for tumor recurrence read an engrossing detective novel during the prescan interval, causing functional activation of Broca’s area and unanticipated increased FDG uptake in this structure (Fig. 23.12). The examples shown in Fig. 23.12 also demonstrate the importance of anatomical-functional fusion images (PET-CT or PET-MRI) to identify structures of interest. FDG is the predominant tumor tracer, but many other PET tracers have been developed to study, characterize, and localize tumors [28].

23.4.4 Functional Imaging Studies of Sensory and Cognitive Activity Reveal Disproportionate Increases in CBF and CMRglc Compared to CMRO2 During Activation

Physiological stimulus and cognitive studies to identify activated brain regions and evaluate interactions among networks of cells during cognitive tasks in human subjects initially relied on PET flow-metabolism assays, but fMRI is increasingly used because exposure to radioactive material is eliminated and repeated studies with high temporal and spatial resolution are possible. For exam-


Fig. 23.12 Localization and characterization of tumors in humans. Left panel: [18F]fluorodeoxyglucose (FDG) localizes metastatic melanoma tumors throughout the body (some are indicated by arrows) that appear as metabolic “hot spots” due to their high glycolytic rates compared to normal tissue. Note that brain has a very high glucose utilization rate compared to other body organs and that the bladder is highly labeled due to clearance of FDG via the kidneys. (Reprinted from [80], with permission from Elsevier). Center panels: a patient with high-grade glioma indicated as a large mass in the MRI image (A, arrow). FDG-PET shows high uptake in the peripheral regions of the tumor (B, arrows) and low uptake in the center of the mass, consistent with a necrotic core. Retention of [18F]fluoromisonidazole (FMISO) varies with oxygen concentration and is much higher under

hypoxic compared to normoxic conditions. The FMISO-PET image shows hypoxic areas (C, arrows) that partially overlap with regions of high FDG uptake. (Reprinted from [81], with permission from Elsevier). Right panels: MRI image (top) and FDGPET/MRI fusion image (bottom) from a patient who was reading a “page-turner” detective novel in the waiting room during the interval between the FDG injection and the PET scan for assessment of metastatic tumors. The FDG-PET shows focal activation in the Broca’s area of cerebral cortex (arrows; high metabolic activity is denoted by color coding of red and white), whereas no anatomical changes were evident in the corresponding MRI images. The interpretation was that reading the exciting novel, not a metastatic tumor, caused local brain activation. (Reprinted from [82], with permission from Lippincott, Williams and Wilkins).

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ple, studies by Raichle and colleagues [83, 84] to evaluate functional metabolic and hemodynamic responses to sensory stimulation and to identify neural correlates of consciousness during skill learning (word processing tasks: speaking, hearing, reading, generating words) revealed brain regions with increased and decreased activity, raising interesting questions regarding factors that govern “baseline” activity itself. One of fascinating findings from activation studies in different laboratories is the apparent dissociation of the stoichiometric relationship between oxygen and glucose utilization in human subjects. Sensory stimulation or mental tasks evoked 30–50% increases in blood flow and CMRglc with little or no change in CMRO2 when assayed by PET [85] or arteriovenous difference [86]. As shown in Fig. 23.13, blood flow and CMRglc rise or fall in parallel during activation or deactivation, respectively, exceeding the much smaller changes in CMRO2 and oxygen extraction fraction (OEF); note that the positive BOLD effect is associated with the small change in CMRO2 and reduced OEF, consistent with a rise in the level of oxyhemoglobin. The CMRO2/CMRglc mismatch is sometimes called “aerobic glycolysis” because it suggests the possibility of increased production and loss of lactate from the activated tissue, even though there are normal and adequate levels and rates of delivery of oxygen. In an animal model for aerobic glycolysis, the changes in brain contents of lactate and glycogen were not sufficient to explain the glucose consumed in excess of oxygen, and analysis of the metabolic fate of [14C]glucose showed that biosynthetic processes could not fully explain the disproportionate utilization of glucose and that glycogenolysis, which occurs in astrocytes, enhances the magnitude of the oxygen-glucose mismatch [87, 88]. Aerobic glycolysis is a very important phenomenon because it reveals a lack of detailed understanding of the loci and identity of energy-producing reactions required to support functional activation. Also, the failure of CMRO2 to equal CMRglc during activation is strong in vivo evidence against a model attempting to explain the cellular basis for activation-dependent increases in CMRglc by linking neuronal excitatory glutamatergic activity to glycolytic metabolism in astrocytes and parallel oxidative metabolism of lactate in neurons via the so-called “astrocyte-to-neuron lactate shuttle”. Some laboratories find that sodium-dependent glutamate uptake stimulates DG metabolism and lactate release from cultured astrocytes (for a review, see [89]), whereas other laboratories find either no effect or an inhibition of CMRglc and lactate release during glutamate exposure; also, glutamate is oxidized by astrocytes and it stimulates oxygen consumption (for reviews, see [90–93]). The basis for these apparently discrepant findings is unknown, but might arise from differences in oxidative capacity in various preparations of cultured astrocytes. Nevertheless, the fact that CMRO2 does not match CMRglc during many activation paradigms in vivo argues against a tight coupling of lactate production, shuttling, and oxidation, since this requires stoichiometric increases in CMRO2 and CMRglc. Thus, if lactate is produced in large amounts during activation, it must be very quickly released from the activated area, since lactate accumulation in brain and oxygen consumption fail to match the rise in glucose metabolism.


Fig. 23.13 Functional activation during visual stimulation. Relationships among blood flow, BOLD response, glucose and oxygen utilization, and oxygen extraction fraction are shown. Visual stimulation with a checkerboard pattern (a) causes an increase in blood flow (CBF) and glucose utilization (CMRglc) in visual cortex, but only a small, if any, rise in oxygen consumption (CMRO2) in this structure, leading to greater oxygen availability in blood because delivery exceeds demand (b). Thus, representative metabolic measurements during brain activation using PET typically show increases in flow and metabolism, but with a disproportionate rise in blood flow and CMRglc compared to CMRO2. This phenomenon is associated

with a smaller oxygen extraction fraction (OEF) and excess oxygen availability in blood, causing a positive fMRI BOLD response (c) (also see Fig. 23.5). In contrast, task-induced reductions in brain activity below the baseline condition of the experimental procedure (deactivation) are associated metabolic changes in the opposite direction (d) compared to activation (c). The basis for deactivation is not understood but is thought to arise from lower overall activity rather than simply increased inhibitory activity, which itself is associated with increased CMRglc. (Reprinted from [84], with permission from the authors and Nature Publishing Group).

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To summarize, unanticipated findings from human sensory and cognitive activation studies using metabolic markers to provide imaging signals exposed major shifts in energy metabolism that are not understood and not explained by current models for astrocyte-neuron interactions. The excess of oxygen availability is registered by metabolic and fMRI BOLD signals; it serves as a strong impetus to understand the biochemical and physiological basis of functional activation.

23.4.5 Imaging Brain Maturation and Aging, Neurotransmitter Systems, and Effects of Drugs of Abuse

Radioactive probes are now available for many purposes, including assay of the activities of specific enzymes in vivo (e.g., hexokinase [CMRglc], thymidine kinase [cell division], aromatic l-amino acid decarboxylase [dopaminergic system], monoamine oxidase, acetylcholinesterase), biosynthetic processes (protein and lipid synthesis), phospholipase-A2-initiated signal transduction activities, neurotransmitter systems (dopamine, serotonin, acetylcholine, GABA, glutamate, opiate) and their components (pre- and postsynaptic structures, rates of neurotransmitter synthesis, transporters, receptors, receptor sub-classes), transporters (sugars, amino acids, monocarboxylic acids, multidrug resistance), abnormal formations (Lewy bodies and b-amyloid), tumors (protein, DNA, and lipid synthesis, receptors, hypoxia, and, angiogenesis, apoptosis, antigens), gene expression and gene therapy, pharmacology, addiction to drugs of abuse, and neurological diseases and disorders [1, 26–28, 94–96]. Disruption of programmed development, damage from drugs of abuse or side-effects of pharmaceuticals, and deficits incurred during aging can all impair brain function; brain imaging techniques can help monitor and evaluate these processes. For example, the maternal-fetal placental barrier permits the passage of FDG [96], and functional metabolic assays in both maternal and fetal organs, including brain, can be carried out in utero (Fig. 23.14), permitting assessment of maturation. Transfer of drugs of abuse, such as [11C]cocaine, from the mother to fetus is also measurable [97]. In aging patients, a battery of assays are available to characterize and quantify specific neurotransmitter systems, as illustrated for the cholinergic system in Fig. 23.14. In dementia patients, deficits in overall “status” (registered by CMRglc) are associated with reductions in vesicular acetylcholine transporters and acetylcholinesterase activity and brain atrophy [98]. The dopaminergic system is involved in the neurobiology of addiction to drugs of abuse, and PET studies help elucidate the actions of drugs of abuse, modulation of reward, motivation, and behavior circuits in brain, and effects of therapeutic interventions. Psychostimulant drugs such as cocaine are associated with blockade of the dopamine transporter, causing an increase in dopamine in the synaptic cleft. In contrast nicotine can stimulate dopaminergic cell firing,


Fig. 23.14 Glucose utilization during fetal development and neurochemical changes in Alzheimer’s disease. Top panels: superimposition of maternalfetal MRI (left two panels) and FDG-PET images (right panel) shows that FDG injected into the maternal circulation of a pregnant monkey labels maternal organs (M-K, kidney; M-I, intestine; M-Bl, bladder), and FDG readily crosses the placenta to produce high labeling of fetal brain (F-Br), forebrain (F-Fb), cerebellum (F-Cb), heart (F-H), and bladder (F-Bl). (Reprinted from [96], with permission from the Society of Nuclear Medicine). Bottom panels: images of statistical maps derived from use of MRI and different PET tracers are used to evaluate anatomical and

neurochemical changes in brain of Alzheimer’s patients, compared to controls of similar age. The highest Z score, denoted by red on the color scale, indicates greatest reduction in glucose utilization (CMRglc) with [18F]FDG-PET, vesicular acetylcholine transporters (VAChT) measured with [125I]iodobenzovesamicol-SPECT, acetylcholinesterase (AChE) activity measured with [11C]methylpiperidyl propionate-PET, and regional atrophy determined by MRI imaging. Most pronounced reductions are in parietotemporal and frontal association cortices, with small or negligible effects in primary sensorimotor cortex and cerebellum. (Reprinted from [98], with permission from Elsevier).

and opiates and alcohol are linked to the disinhibition of dopaminergic cells [99]. Specific PET tracers, such as [11C]raclopride that bind to dopamine receptors can reveal relationships between the perceived drug-induced “high”, occupancy of the receptor, and overall functions (assayed with [18F]FDG) of the brain circuits implicated in addition (Fig. 23.15). One of the major mechanisms for inactivation of catecholamine neurotransmitters is oxidation by monoamine oxidase (MAO). Recent studies with a PET tracer that measures the amount of

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Fig. 23.15 Brain circuits involved in drug abuse in human addicts. Top panels: (A) When DA is released from the synaptic terminal into the cleft, it can bind to the D2 receptor on the postsynaptic neuron, or be taken up back into the presynaptic neuron via a DA transporter (DAT). [11C]Raclopride can bind to D2 receptors only if they are not occupied by dopamine (DA; cartoon top left). Methylphenidate (MP) is a stimulant drug that blocks DA transporters causing DA to accumulate in the synaptic cleft, thereby reducing the number of

unoccupied receptors and inhibiting binding of [11C]raclopride (cartoon top right). The two brain images show that [11C]raclopride binding is higher after placebo treatment (bottom left) and reduced after MP treatment (bottom right), consistent with higherthan-normal amounts of DA in MP-treated subjects. (B) The higher the MP-induced rise in DA (expressed as Bmax/Kd from [11C]raclopride assays), the greater the magnitude of the self-reported MP-induced “high” or sense of euphoria in nonaddicted subjects.


Fig. 23.16 Enzyme-catalyzed covalent binding of [11C]substrate quantifies enzyme amount. MAO A and B are an important enzymes that degrade bioactive amines, including catecholamines. Selective metabolism of L-deprenyl by MAO B results in stoichiometric, covalent binding of deprenyl to the enzyme; [11C]deprenyl can therefore be used to visualize and quantify the concentrations of MAO B in body organs. Whole-body PET scans after injection

of [11C]deprenyl show marked, generalized depression of MAO B level in body organs of smokers who consumed an average of about 25 cigarettes per day for 22 years. Color scales are equivalent in the representative nonsmoker and smoker subjects: red is the highest 11C concentration. (Reprinted from [100], with permission from the United States National Academy of Sciences).

Fig. 23.15 (continued) Bottom panels: cocaine also blocks the DA transporter. Addicted cocaine abusers have decreases in DA D2 receptors in the striatum (top left), reflected by lower binding of [11C]raclopride. The addicts also have a blunted response to MP administration, suggesting two abnormalities in these subjects: fewer D2 receptors and reduced DA release. Cocaine abusers also have reduced rates of glucose utilization in orbito-

frontal cortex (bottom left), suggesting subnormal brain function. The graphs in the right panels show the correlations between D2 receptor availability and CMRglc in detoxified cocaine and metamphetamine abusers: the lower the D2 receptor availability, the lower the CMRglc. The orbitofrontal cortex and cingulated gyrus are thought to be motivational/drive circuits that are disrupted in drug abusers. (Reprinted from [99], with permission from Elsevier).

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MAO B (one of the two isoforms of MAO) show a dramatic reduction in the level of this enzyme throughout the body of chronic smokers (Fig. 23.16). This effect is not related to nicotine, indicating that tobacco smoke has widespread effects on many different organs [100]. These examples illustrate the use of PET imaging to investigate specific neurotransmitter systems at the molecular, cellular, and organ level in living subjects.

23.4.6 Imaging Electrolyte Transport Across the Blood-Brain Interface and Shifts in Calcium Homeostasis Under Pathophysiological Conditions

Transport of electrolytes across the blood-brain interface is very restricted, compared to the rapid, high-capacity transport of glucose. The highest rate of transfer electrolytes from blood to brain is across the choroid plexus; it is less extensive across arachnoid-pial membranes, and quite low across endothelial cells. Autoradiographic analysis of blood-to-brain transfer of a representative cation, 45 Ca2+, shows highest labeling of periventricular tissue at 1 h after a pulse intravenous injection of tracer; by 5 h the 45Ca2+ distributes widely within brain, followed by clearance at 24 h (Fig. 23.17) [101]. Studies with other radiolabeled electrolytes, including Na+, K+, Mg2+, Mn2+, Cl–, and PO2– 4 , show similar labeling patterns (see [102] and references therein). Brain regions vulnerable to transient ischemia exhibit abnormal calcium homeostasis that becomes manifest along with the evolution of ischemic cell change. Increased uptake and trapping of calcium and greater entry of bloodborne calcium into tissue compartments not labeled in normal tissue is readily detected and quantified by 45Ca autoradiography after transient ischemia (Fig. 23.17). Accumulation of blood-borne 45Ca in the ischemic-damaged choroid plexus is evident at 2–5 h after onset of reperfusion, in the caudate within 5 h, and at 24–72 h in the CA1 hippocampus, consistent with delayed onset of ischemic damage in the hippocampus compared to caudate. Local secondary calcium responses that occur after a primary excitotoxic challenge of hippocampal neurons in brain slices with kainic acid are readily detected by optical imaging using a calcium-binding fluorescent dye [103]. These calcium responses originate in dendrites, and over a period of minutes, spread to involve cell bodies, then affect the entire cell and eventually lead to death (Fig. 23.17). These results illustrate an advantage of combining in vivo and in vitro studies because temporal profiles of regional disruption of calcium homeostasis can be identified, and detailed analysis of the dynamics and mechanisms of calcium overloading can be assessed. Control of hydrogen ion concentration is essential for normal cell function. For example, regulated transfer of protons across mitochondrial membranes are used to generate energy, and proton-coupled transport of metabolites, such as facilitated cotransport of H+ plus lactate by the MCT, can clear metabolic byproducts and help control intracellular pH during functional activation or tissue


Fig. 23.17 Calcium homeostasis in normal and ischemic-damaged brain and during excitotoxic challenge. Left two columns: top row is hematoxylin and eosin-stained coronal sections of rat brain at the level of the anterior hippocampus (A) and caudate (B). Cx = cerebral cortex; H = hippocampus; T = thalamus; V = lateral ventricle that contains choroid plexus (CP); cc = corpus callosum; c = caudate; ac = anterior commisure. 45Ca autoradiographs prepared from normal rats at the indicated intervals after a pulse intravenous injection of 45Ca show entry of calcium into brain mainly via the choroid plexus (1 h), spreading within brain to produce more uniform labeling of the brain at 5 h, and clearance from brain at 24 h. Middle two columns: rats subjected to 30 min of complete forebrain ischemia by four-vessel occlusion incur ischemic damage that becomes maximal in the caudate within 24 h and is delayed in the hippocampus, becoming evident at 24 h and maximal at 72 h. When 45Ca is injected immediately after onset of reperfusion there is intense labeling of the choroid plexus, which is damaged by ischemia (1–5 h; A, B),

increased levels of 45Ca in the ischemicdamaged CA1 hippocampus at 24 h and increasing at 72 h (A); labeling of the dendritic regions is most prominent. In contrast, accumulation of 45Ca in the ischemicdamaged caudate is evident within 5 h and high levels persist at 24–72 h. Note that 45 Ca is gradually cleared from undamaged regions in the rats subjected to ischemia (e.g., cerebral cortex and thalamus; A, B). (Reprinted from [101], with permission from Blackwell Publishing). Right panels: the fluorescent calcium indicator dye reveals local Ca2+ responses in hippocampal dendrites in mouse brain slices exposed to kainic acid. Within 3 min after washout of the kainic acid, dendrites recovered to resting Ca2+ levels (a), but shortly thereafter, secondary Ca2+ responses were evident in dendrites (b), followed by propagation into secondary dendrites (c–d), before reaching (e) and involving the soma (f). Cascades leading to elevated levels of Ca2+ can involve the entire cell and lead to neuronal death. (Reprinted from [103], with permission from The Society for Neuroscience).

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Fig. 23.18 Rapid waves of stimulus-induced spreading acidification. (A) Neutral red, a pH-sensitive dye was administered before the stimulus session. Color-coded images show spreading acidification in rat cerebellar cortex induced by a 10-s pulse train superimposed on the surface image of the brain. The first image (top left) is a background image showing the electrode position; the yellow line indicates the plane of the parasagittal section shown in (B).

The series of images shows the progressive spreading of acidification across the tissue; the numbers in the upper right corner of each successive image indicates the time (s) after stimulus onset. The distance of spreading at four time-intervals is shown in (C); and (D) shows that the rise in DF/F corresponds to a fall of about 0.3 pH units, as measured with a pH-sensitive electrode. (Reprinted from [35], with permission from Sage Publications).

injury that increases glycolytic metabolism. pH-sensitive fluorescent dyes have been used to image pH changes in tissue slices from funnel-frozen brain (Fig. 23.11) and living brain (Fig. 23.18). Stimulus-induced changes in neutral red fluorescence reveal a rapidly spreading wave of acidification associated with depression of pre- and postsynaptic neuronal activity. The acid-shift is about 0.2 pH units, and it spreads across the cerebellar surface at about 500 lm s–1, peaking at twice that speed; these rates are about ten times greater than those of spreading cortical depression or calcium waves. The phenomenon of spreading acidification depends on intact circuitry and can be modulated by various interventions, but the mechanisms of acid production and neuronal depression remain to be clarified [35]. Given the general tendency for greater utilization of glucose compared to oxygen during activation (Section 23.4.4), it is tempting to speculate that spreading acidification waves might involve, at least in part, lactate formation and transport.

23.5 Optical Imaging of Functional Activity by Means of Fluorescent Compounds

23.4.7 Summary

Molecular imaging probes are used to assess a broad spectrum of biological processes and functions ranging from enzyme activities to transmitter systems and complex cognitive processing. These techniques have revealed unexpected and poorly understood phenomena (aerobic glycolysis, BOLD contrast, and spreading acidification) that are now stimulating more studies designed to better understand the cellular interactions and regulatory processes involved in relationships among blood flow, metabolism, and function.

23.5 Optical Imaging of Functional Activity by Means of Extrinsic and Intrinsic Fluorescent Compounds

The use of voltage-sensitive dyes to register and report very fast changes in membrane potential has made enormous progress in the past decade, and the approach has great promise for future studies of membrane-related events linked to cortical dynamics and sensory perception [2]. Optical imaging has the advantage of high spatial resolution (50 lm), and assays can now be carried out in conscious monkeys during behavioral tasks, with reproducible results in studies extending over periods approaching a year (Fig. 23.19). The voltage-sensitive dyes bind to the exterior membranes and have millisecond time resolution; the amplitude of the signals is directly proportional to membrane voltage and area, corresponding to electrical activities of cells [2]. These properties provide advantages over assays based on hemodynamic changes that are indirect measures of cellular activity plus blood flow responses. This technology has applications in multimodal studies, and will be useful in analysis of single cell and multicellular activities during perception, behavior, learning, memory, plasticity, and other complex, higher brain functions [2]. The analysis of intrinsic signals derived from autofluorescence of NADH plus NADPH and oxidized flavoproteins is an emerging field that shows considerable promise in helping to understand the cellular and subcellular roles of glycolytic and oxidative metabolism during functional activation [4–6]. For example, sensory stimulation of the anesthetized rat caused an increase in flavoprotein fluorescence (Fig. 23.19) that had the same distribution as cortical field potentials; and the oxidative metabolic change was followed by an increase in blood flow that could be attenuated by nitric oxide synthase inhibitor, whereas the fluorescence signal was not altered. The combined use of flavoprotein autofluorescence to evaluate oxidative metabolism and laser speckle imaging to assess local changes in blood flow showed a rapid increase in oxidative metabolism in rat whisker barrel cortex, with a lag of about 100 ms after onset of vibrissa stimulation in anesthetized rats. In contrast, the blood flow response was slower, rising at 600 ms and peaking at about 1.4 s after stimulus onset and involving about

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Fig. 23.19 Optical imaging of changes in membrane potential and cellular redox state during functional activation. Left panels: top figure illustrates an in vivo system for chronic optical imaging of cortical dynamics in conscious monkey during visual stimulation, using voltage sensitive dyes. The exposed cortical tissue is covered with a sealed chamber (inset, lower left) and the cortex is stained with a suitable dye and illuminated with an external light source so that serial fluorescent images can be captured with high temporal (milliseconds) and spatial (~200 lm) resolution during visual stimulation, digitized, analyzed, and displayed as functional maps. Electrical recording along with the optical imaging shows congruence of the intracellular activity (blue) and voltage-sensitive dye population activity (red; inset, middle right). The lower figure illustrates the area of exposed cortex in the sealed chamber (a) and the functional voltage-sensitive dye imaging maps (b) of ocular dominance (left and right) and orientation (middle) recorded from the same

monkey in recording sessions about a year apart (compare top and bottom panels). (Reprinted from [2], with permission from the authors, Nature Publishing Group, and Springer). Right panels: autofluorescence of mitochondrial flavoproteins changes in response to vibratory stimulation of the skin of the rat forepaw, revealing changes in redox state: responses to (A) forepaw stimulation, (B) a single forepaw stimulus trial, (C) hindpaw stimulation. The brain surface image is shown in (D). The cresyl violetstained parasagittal section of cortex (E) indicates the boundary between motor and somatosensory cortices (black arrow) and an electrolytic lesion (red arrow) made at the center of the autofluorescence responses in (C). Autofluorescence responses to 50 Hz, 1 s hindpaw stimulation and field potentials (G) were elicited by a single 10-ms stimulus and recorded at the colored spots in F: colors identify corresponding spots and traces. (Reprinted from [4], with permission from the authors and Blackwell Publishing).

23.6 Tracking Dynamic Movement of Cellular Processes and Cell Types

2.5 times more tissue than metabolic activation [104]. In cerebellar cortex of anesthetized mice, surface stimulation evoked a flavoprotein-derived fluorescence signal that was linearly related to the stimulus amplitude, frequency, and duration. Also, the signal could be reduced by impairment of mitochondrial respiration with cyanide without altering the pre- and postsynaptic components of the electrophysiological response [6]. In hippocampal slices, the stimulus-induced rise in NAD(P)H fluorescence was an “inverted match” of the FAD fluorescence; and it was attributed mainly to postsynaptic neuronal activity because it was abolished by ionotropic glutamate receptor block [5]. Taken together, studies of autofluorescence signals derived from intrinsic molecules should help understand the cellular basis of shifts in glycolytic and mitochondrial metabolism as neurons and astrocytes respond to various stimuli.

23.6 Tracking Dynamic Movement of Cellular Processes and Cell Types

Synaptic remodeling during development and cellular responses to brain injury are associated with the movement of subcellular structures and migration of specific cell types within brain. For example, the mobility of subcellular components and dynamic movement of synaptic spines are essential for plasticity and reorganization. Exposure of cultured astrocytes to the excitatory neurotransmitter glutamate causes filopodial extension and elongation within 30 s [105]; spontaneous movement of astrocytic fine processes in the territory of active synapses is visualized in brain slices of transgenic mice that express high levels of green fluorescent protein. Astrocytes exhibit the gliding type of movement and process extension that takes place over periods of minutes (Fig. 23.20). The significance of astrocytic process mobility remains to be established, but it raises the fascinating possibility that interactions among brain cells might involve temporary changes in proximity to better control synaptic activity and signaling strength, perhaps by regulating glutamate uptake into astrocytes [36, 105]. Macrophages and microglia “patrol” the brain and contribute to inflammatory responses after brain infection and injury. These cell types express peripheraltype benzodiazepine-binding sites that selectively bind the ligand PK 11195; macrophages and microglia responding to ischemic damage can be localized and quantified in animals and human patients with 3H- and 11C-labeled PK 11195 [106, 107]. [11C]PK 11195 has also been used to identify brain regions involved in multiple system atrophy [108]. Noninvasive assay of cellular migration within brain can also be carried out by using ultrasmall superparamagnetic iron oxide (USPIO) as an MRI contrast agent [109, 110], and movement of USPIOlabeled macrophages and stem cells into the territory of ischemic damage can be readily visualized and tracked over periods of days (Fig. 23.21). Thus, new strategies involving radiolabeled ligands that are specific for cell types or prelabeling cells with NMR contrast agents enable the in vivo analysis of cellular movements.

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Fig. 23.20 Dynamic mobility of astrocytic filopodia near active synaptic terminals. Transient extension of filopodia (a) in an acute brain slice preparation from a transgenic mouse that expresses green fluorescent protein in astrocytes is visualized in time-lapse images taken in a single plane at 2-min intervals. (b) 3-D reconstruction of time-lapse images after deconvolution. (c) Temporal profile of filopodial movement, expressed as volume changes determined

from the reconstructed 3-D images. The astrocyte processes are in close proximity to active synapses identified by uptake of FMI-43, a fluorescent dye that is taken up during recycling of synaptic vesicles. Two types of astrocyte process movement were identified, lamellipodia-like membrane gliding movements and filopodial process extensions. (Reprinted from [36], with permission from Blackwell Publishing).

23.7 Evaluation of Exogenous Genes, Cells, and Therapeutic Efficacy

Movement disorders associated with Parkinson’s disease arise from the slow, progressive degeneration of dopaminergic neurons in the substantial nigra. Nigral cell death causes depletion of dopamine levels in the striatum and gradual acquisition of motor symptoms, tremor, disruption of gait, and other complications that become evident when dopamine levels are reduced to about 20–40%


Fig. 23.21 Tracking cellular movements within brain after ischemic injury. Top panels: macrophages in damaged brain are visualized by NMR by means of ultrasmall superparamagnetic iron oxide (USPIO)-containing particles that are engulfed by the macrophages. At 5 days after photothrombolytic infarction, rats were injected with USPIO (A) or saline (B) and imaged 24 h later by MRI, to reveal a rim of hypointensity (red arrows, A) around the zone of infarction characterized by signal loss on T2*-weighted images. The middle region of the infarct remained hyperintense, as did the entire infarcted region in the saline-injected rat (white arrows, A, B). The USPIO contrast image coincided with those obtained by histochemical detected iron and ED1 immunohistochemical assay of macrophages (not shown). (Reprinted and modified from [109], with permission from John Wiley and Sons). Bottom panels: embryonic stem cells

labeled with USPIO using a lipofection technique were injected into two sites (ventral cortical layer near the border of the corpus callosum [green arrow] and in the caudate [yellow arrow]) 2 weeks after induction of transient focal ischemia in the contralateral hemisphere by 60-min occlusion of the middle cerebral artery. MRI imaging shows loss of contrast (dark areas indicated by arrows) at the injected sites on the day of the injection (A) and 6 days (B) or 8 days (C) later. Migration of stem cells is shown (red arrows) along the corpus callosum (B, and at higher magnification in D, arrow) and the ventricular wall (E) and to the choroid plexus (E). The necrotic zone is outlined in the right hemisphere in (C); a dark region in the dorsal part of the damaged territory reflects the arrival of the USPIO-labeled stem cells. (Reprinted and modified from [110], with permission from the United States National Academy of Sciences).

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of normal. Because dopamine cannot cross the blood-brain barrier, neurotransmitter replacement therapy involves the treatment of Parkinson’s patients with the precursor, l-dopa. Dopa crosses the blood-brain interface and is decarboxylated in brain by aromatic l-amino acid decarboxylase (AADC) to form dopamine that can then interact with postsynaptic receptors in the striatum and ameliorate dysfunction. Unfortunately, this treatment paradigm has side-effects, and with time, its efficacy diminishes due, in part, to progressive loss of the decarboxylase as the presynaptic neurons die. Alternative therapeutic approaches, including gene replacement via viral vectors or cell injection, have been tested in animal models for Parkinson’s disease produced by treatment with MPTP. This toxin selectively damages dopaminergic neurons and causes motor disturbances. The efficacy of a viral vector system to incorporate AADC into the striatum of an MPTP-treated monkey is shown in Fig. 23.22. Functioning of the AADC gene after its incorporation into brain tissue was visualized by PET using [18F]fluoro-l-m-tyrosine (FMT), a substrate for AADC: the enzymatic product is a tyramine derivative that accumulates in dopaminergic terminals and serves as a measure of dopamine synthesis capacity [111]. A different approach to help restore striatal function of MPTP-treated monkeys is implantation of human retinal pigment epithelial cells that have the capability to produce l-dopa [112]. PET imaging with [18F]fluorodopa showed that cell-implant therapy enhanced AADC activity and increased the rate of formation of fluorodopamine that is retained in presynaptic terminals; also, receptor binding of [11C]raclopride fell after treatment, consistent with increased competition of the PET tracer with higher endogenous dopamine levels (Fig. 23.22). Thus, pre- and postsynaptic activity of the dopaminergic system can be assessed in vivo and correlated with behavioral improvement after therapeutic intervention. In vivo assessment of tumor cell proliferation and response to gene therapy treatment employs genetic engineering and various imaging strategies involving optical, NMR, and PET modalities [113]. For example, insertion of the luciferase gene into mice allows the monitoring of tumor growth in brain in vivo (Fig. 23.22). The quantity of light emitted has been shown to be proportion to tumor size, and longitudinal imaging of tumor response to treatment via different signaling pathways can be followed in individual animals, thereby circumventing problems associated with inherent variability arising from initial differences in tumor size and depth [114]. Another approach to monitoring gene therapy is to incorporate an engineered reporter gene into the system so that when the gene is expressed and translated, the reporter protein can be detected by

Fig. 23.22 Imaging activities of exogenous genes and cells in brain. (A) PET imaging of enzyme replacement therapy by means of gene restoration or cell implantation. The top panels show imaging of a monkey previously treated by unilateral

infusion via the right internal carotid artery of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), a dopaminergic toxin that causes clinical signs of Parkinson’s disease. Partial loss of dopamine-producing cells in Parkinson’s patients is treated by providing


Fig. 23.22 (continued) the precursor, L-DOPA, that can cross the blood-brain barrier; one approach is to restore aromatic L-amino acid decarboxylase (AADC) activity so that the enzyme can synthesize dopamine from peripherally administered precursor. Before gene therapy with a viral vector containing the AADC gene (left panel), the monkey was imaged with an AADC tracer, 6-[18F]fluoro-L-m-tyrosine (FMT), that showed low FMT uptake in the MPTP-treated (right) striatum (arrow). After gene therapy (right panel), AADC activity increased, causing a large rise in FMT uptake in the treated (right) striatum (arrow). (Reprinted from [111], with permission from Academic Press). The bottom two panels show an alternative approach, i.e., direct injection of human retinal pigment epithelial cells (hRPE) attached to gelatin microcarriers into the putamen of bilaterally MPTP-treated monkeys; hRPE cells produce L-DOPA, the precursor for dopamine that would be produced by residual dopaminergic neurons. Imaging of the effects of these cells with L-[18F]fluoroDOPA (tracer for decarboxylation to fluorodopamine and storage in presynaptic dopamine

terminals) shows increased uptake in the hRPE-injected putamen (arrow, left panel) and decreased binding of [11C]raclopride, consistent with higher endogenous production of DOPA that would bind to the dopamine receptors, inhibiting binding of [11C]raclopride in the hRPE-treated putamen (arrow) compared to the contralateral structure (also see Fig. 23.15). (Reprinted from [112], with permission from Academic Press). (B) Bioluminescence imaging of tumor growth and regression during treatment. Top panels show that that platelet-derived growth factor (PDGF)-driven oligodendrogliomas induced in transgenic mice expressing the luciferase gene emit more light (top two panels, a) when tumor size is larger, shown by histological staining (b). The graph in the lower panel shows light emitted from control mice and from an untreated tumor-bearing mouse, followed by blockade of the PDGF receptor causing tumor growth cycle arrest and reduction of tumor size and bioluminescence in serial assays carried out over a 6-day period. (Reprinted from [114], with permission from the authors and Nature Publishing Group).

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PET or NMR imaging. Imaging depends upon: (1) binding to a suitable radiolabeled or paramagnetic reporter probe, (2) transport, or (3) metabolism of the probe by the reporter protein so that the probe or its labeled product is trapped in the cells where the gene is expressed. For example, transfection of a viral thymidine kinase gene into tumors is an experimental therapy because a prodrug (e.g., ganciclovir) can be administered systemically, taken up, and trapped by phosphorylation by the viral thymidine kinase, followed by further phosphorylation by cellular enzymes and incorporation into tumor DNA where it acts as a chain terminator to kill the tumor cells. Expression of the enzyme can be detected by PET assays of 18F-labeled reporter probes that are taken up and phosphorylated [115, 116]. Clever applications of genetic engineering coupled with brain imaging techniques using unique reporter systems and pharmaceuticals will enable both direct and indirect assessment of disease states and novel therapeutic advances. 23.8 Summary and Perspectives

The physical structure and biochemical composition of brain are designed to support and maintain a large and very complex range of functional activities by different types of cells in brain. To visualize and quantify biological processes in living brain, neuroscientists must take into account the properties of the bloodbrain interface and the regional complexity and heterogeneity of the brain. The basic principles and methods first devised for the assay of local rates of blood flow and glucose utilization have been widely applied, refined, and extended. During the past 40 years, novel new technologies, extraordinarily useful probes, and insightful experimental strategies have been developed for molecular imaging to localize and quantify biological processes ranging from gene expression, specific enzymatic reactions, signaling activities and pathways, metabolite and electrolyte transport, concentrations and redox state of key components of blood and brain, cellular movement, and complex higher functions of brain in living organisms. Different imaging modalities have advantages and disadvantages in terms of temporal and spatial resolution, single or repeated assays in longitudinal studies, cost and complexity, and types of applications. The fusion of anatomical and functional images derived from extrinsic and intrinsic reporter molecules and multimodal approaches are essential for the analysis of dynamic aspects of brain function. Challenges for the future include understanding the biochemical and physiological basis for many of the key imaging signals and neurological and mental diseases, and developing effective treatments for brain disorders and tumors.

Acknowledgments

Supported by NIH grants NS36728 and NS38230.

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Part VII Disease-Related Response


603

24 Inflammatory Response of the Blood-Brain Interface Pedro M. Faustmann and Claus G. Haase

24.1 Introduction

The central nervous system (CNS) is considered an immunoprivileged site where the blood-brain barrier (BBB) controls cellular and serum protein entry into the brain. Under pathological conditions, such as inflammation, trauma, and neurodegeneration, a large number of white blood cells (WBCs) immigrate into the CNS. Additionally, changes of protein concentrations in the cerebrospinal fluid (CSF) indicate a disruption of the BBB. The mechanisms allowing WBCs to pass the BBB, and which protein in the CSF is disease-related represent central questions in neurology, neuropathology, and neuroimmunology. Obviously, they are important for our understanding of the pathogenesis of inflammatory brain diseases.

24.2 Diagnostic Features of Cerebrospinal Fluid

Normal CSF is crystal clear. However, as few as 200 WBCs mm–3 or 400 red blood cells (RBCs) mm–3 cause CSF to appear turbid. Xanthochromia (yellow-colored) is most often caused by lysis of RBCs, resulting in hemoglobin leakage and breakdown to oxyhemoglobin, methemoglobin, and bilirubin. CSF protein levels of at least 150 mg dl–1 (1.5 g l–1), as seen under infectious and inflammatory conditions, or as a result of a traumatic tap that contains more than 100 000 RBCs mm–3, also results in xanthochromia. Newborn CSF is often xanthochromic because of the frequent elevation of bilirubin and protein levels.


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24 Inflammatory Response of the Blood-Brain Interface

24.2.1 Cell Count and Cell Differentiation

Normal CSF may contain up to 5 WBCs mm–3 in adults and 20 WBCs mm–3 in newborns. About 90% of patients with bacterial meningitis have a WBC count higher than 1000 cells mm–3 while 99% have more than 100 cells mm–3. Less than 100 WBCs mm–3 is more common in patients with viral meningitis. Elevated WBC counts (> 20, < 100) may also occur after seizure, in intracerebral hemorrhage, brain tumors, and under a variety of inflammatory conditions. Table 24.1 lists common CSF parameters under various types of meningitis. The WBC count seen in normal adult CSF is comprised of approximately 70% lymphocytes and 30% monocytes. The majority of patients with polyradiculitis will have < 10 monocytes mm–3. Up to 50 monocytes mm–3 are seen in about 25% of patients with multiple sclerosis. The cell composition of CSF does not allow to differentiate between bacterial and nonbacterial meningitis. For instance, lymphocytosis is frequently seen in viral, fungal, and tuberculous infections of the CNS, although a predominance of polymorphonuclear leukocytes (PMN) may be present in the early stages of these infections. CSF in bacterial meningitis is typically dominated by the presence of PMNs. However, more than 10% of cases of bacterial meningitis shows a lymphocytic predominance, especially at early stages of the clinical course and when the number of WBCs are less than 1000 cells mm–3. Eosinophilic meningitis is characterized by > 10 eosinophils mm–3 or, when a total CSF cell count is made, > 10% eosinophils, a feature that is suspective for a parasitic infection. Other possible causes for eosinophilic CSF may include viral, fungal, or rickettsial meningitis; ventriculoperitoneal shunts with or without coexisting infection; malignancy; and adverse drug reactions.

Table 24.1 Cerebrospinal fluid findings in meningitis. Test

Bacterial

Viral

Fungal

Tubercular

White blood cell count

³ 1000 per mm3

< 100 per mm3

Variable

Variable

Cell differential

Predominance of PMNs

Predominance of lymphocytes



Protein

Mild to marked elevation

Normal to elevated

Elevated

Elevated

CSF : serum glucose ratio

Normal to Usually normal marked decrease

Low

Low

24.3 Acute Bacterial Meningitis

24.2.2 Protein Level

CSF protein concentration is one of the most sensitive indicators for a pathology in the CNS. Newborns show up to 150 mg dl–1 (1.5 g l–1) of protein. The adult range is 18–58 mg dl–1 (0.18–0.58 g l–1). The adult level is achieved between six and 12 months of age. Elevated CSF protein levels are seen in infections, intracranial hemorrhages, multiple sclerosis, Guillain Barré syndrome (polyradiculitis), malignancies, some endocrine abnormalities, certain medication, and a variety of inflammatory conditions (Table 24.2). Protein concentration is falsely elevated by the presence of RBCs in a traumatic tap situation.

24.2.3 Glucose Level

CSF glucose renders about two-thirds of serum glucose. This ratio decreases with increasing serum glucose levels. CSF glucose levels generally do not exceed 300 mg dl–1 (16.7 mmol l–1) regardless of serum levels (see Chapter 4). CNS infections can cause lowered CSF glucose levels, although glucose levels are usually normal under viral infections. Normal glucose levels do not rule out infection. Up to 50% of patients who have bacterial meningitis have normal CSF glucose levels. Chemical meningitis, inflammatory conditions, subarachnoid hemorrhage, and hypoglycaemia may also be responsible for hypoglycorrhachia (low glucose level in CSF) [1–6].


More than 50 years after the advent of antibiotic therapies, the mortality and rates of morbidity associated with bacterial meningitis remain high. Meningitis due to Streptococcus pneumoniae has the highest fatality rate of about 20–30%,

Table 24.2 Average and range of cerebrospinal fluid protein. Condition

Average: mg dl–1 (g l–1)

Average: mg dl–1 (g l–1)

Bacterial meningitis Brain tumor Brain abscess Aseptic meningitis Cerebral hemorrhage Neurosyphilis

418 115 69 77 270 68

21–2220 (0.21–22.2) 15–1920 (0.15–19.2) 16–288 (0.16–2.88) 11–400 (0.11–4.0) 19–2110 (0.19–21.1) 15–4200 (0.15–42.0)

(4.18) (1.15) (0.69) (0.77) (2.7) (0.68)

605

606


and up to one-third of survivors are left with significant disabilities or handicaps [7]. The unfavorable clinical outcome of bacterial meningitis is often due to intracranial and cerebral complications comprising cerebrovascular insults, vasogenic and cytotoxic brain oedema, hydrocephalus, and increased intracranial pressure. An overshooting immune response of the host is thought to be responsible for brain damage, which may result in neurological sequelae including hearing loss, symptomatic epilepsy, focal neurological deficits, and mental retardation. Experimental studies using animal models and cell culture systems have substantially increased our knowledge of the pathogenesis and pathophysiological mechanisms during bacterial meningitis. Acute breakdown of the blood-brain barrier, accumulation of PMN in the CSF, and intrathecal production of cytokines are key features in disease development which have proven fundamental for brain oedema, cerebral vasculitis, brain ischemia, and ultimately for neuronal cell death [8–11]. The clinical diagnosis of bacterial meningitis is established by identification of the bacterial microorganism in the CSF and by a high number of CSF cells (> 1000 leukocytes ll–1) consisting of more than 60% PMNs [1].

24.3.1 Bacteria and the Blood-Brain Barrier

Meningitis caused by Haemophilus influenzae type b in children has been eliminated due to routine immunization of infants against this pathogen. This fact has favored the preponderance of S. pneumoniae and Neisseria meningitidis as the most prominent pathogen of meningitis in young children; and meningitis by H. influenzae type b is now a disease predominantly occurring in adults. Among neonates, S. agalactiae, Escherichia coli, and Listeria monocytogenes are the leading pathogens of bacterial meningitis. Bacteria enter the CNS via the vasculature or through focal infections located close to the CNS. Bacterial binding to the cerebral microvascular endothelium is considered an important initial step in the pathogenesis of meningitis. In vitro models of bacterial meningitis have been used to study the interaction of bacteria and bacterial components with cultured cerebral microvascular endothelial cells, but the exact interactions are still incompletely understood. We will discuss a few of them in the following [12]. Invasion of S. pneumoniae into an endothelial cell layer is dependent on the presence of pneumococcal choline-binding protein A, and invasion is partially inhibited by antagonists to the platelet-activating factor (PAF) receptor on the endothelial cell membranes [13]. Pneumococcal surface protein A (PspA) is regarded as an important virulence factor and has been found to interfere with complement activation [14]. Additionally, it was shown that pneumococci expressing pneumolysin were able to breach the endothelial cells, whereas mutant pneumococci deficient in pneumolysin were unable to penetrate the cell barrier [15]. Pneumolysin is a 53-kDa protein, lytic to all cells with cholesterol-contain-


ing membranes. Pneumolysin has been shown a key virulence factor which seems to be responsible for the high morbidity and mortality of pneumococci infections [16]. Whereas pneumolysin inhibits neutrophilic and monocyte function, it also induces the production of proinflammatory mediators, such as tumor necrosis factor alpha (TNF-a), interleukin-1 (IL-1), and IL-6 [17]. For N. meningitidis, surface structures of type IV pili (a bacterial appendage) have been identified as being essential for meningeal invasion [18]. The pili interact with CD46, a human cell-surface protein involved in the regulation of complement activation [19]. Additionally, fibronectin has been identified as an uptake-promoting serum factor, which binds to human brain-derived endothelial cells via integrin receptors [20]. E. coli enter the endothelial cell of the BBB by interaction of various bacterial proteins with endothelial receptors [21]. Within the CSF, bacteria multiply, lyse spontaneously and release proinflammatory and toxic compounds by autolysis and secretion. Lipopolysaccharide (LPS) is derived from bacterial cell walls and affects the passage of other proteins across the blood-brain barrier through the release of cytokines and disruption of the BBB [22, 23].

24.3.2 Leukocyte Migration into the CNS

Under normal conditions, the concentrations of leukocytes, complement factors, and antibodies in the CSF are low. PMNs are attracted into the subarachnoid space by proinflammatory mediators which are initially produced by meningeal and perivascular macrophages [24] and later by immigrated PMNs and local microglia. Generally, the high influx of PMNs into the subarachnoid space is associated with the degree of neurological damage in patients with bacterial meningitis. However, the effects of PMNs in the CSF are dependent on the bacterial titer in the CSF and the clinical time-course of the meningitis. An initial high CSF-PMN count prior to antibiotic therapy is associated with a greater chance of survival, suggesting suppression of bacterial multiplication by the PMNs. A continuous CSF-PMN invasion during antibiosis is associated with a greater chance of death, indicating detrimental effects of CSF pleocytosis in the context of effective therapy [25]. Several experimental studies suggest two strategies to decrease PMNs immigration into the CSF: (a) inhibition of adhesion of PMNs to the luminal endothelial surface, and (b) inhibition of attraction (chemotaxis) of PMNs to the subarachnoid space. PMN adhesion to the endothelial surface is initially mediated by the interaction of endothelial selectins with CD15 on the leukocyte surface inducing the phenomenon of leukocytic rolling. Selectinmediated rolling of PMNs is essential for the transendothelial migration of PMNs, as was shown in selectin-deficient mice [26]. Inhibition of PMNs rolling by a selectin-blocker, the polysaccharide fucoidin, attenuates the inflammatory response in experimental pneumococcal meningitis [27, 28]. Interestingly, an-

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other polysaccharide, glucuronoxylomannan (GXM), a capsular component of the fungus Cryptococcus neoformans, delays translocation of PMNs across the BBB in an animal model of acute bacterial meningitis [29]. A similar inhibitory effect on PMN adhesion is observed when antibodies directed against the intercellular adhesion molecule (ICAM)-1 [30] are used in experimental meningitis [31]. Transgenic deletion of ICAM-1 led to a more than 50% reduction in the number of infiltrating PMNs in an experimental model of inflammation using systemic lipopolysaccharide (LPS) injection [32]. Antibodies against the junctional adhesion molecule (JAM), a cerebral endothelial tight junction component, inhibit PMN extravasation only in a model of cytokineinduced meningitis in mice [33], but not in a model of infectious meningitis. Moreover, anti-JAM antibodies induce a complement-mediated damage of the endothelium [34]. The process of PMN attraction to the subarachnoid space is regulated by chemotactic substances such as complement factor C5a [35–37], platelet-activating factor, and chemotactic cytokines called chemokines. Chemokines are produced at sites of inflammation and subsequently presented at the luminal side of the endothelium of the postcapillary venules, sometimes in cooperation with surface heparin sulfates [38]. Increased concentrations of interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory proteins MIP-1a and MIP-1b are found in the CSF of patients with bacterial meningitis [39, 40]. In experimental models of meningitis, PMN infiltration of the subarachnoid space is only slightly or not at all diminished by intracisternal administration of anti-IL-8 antibodies, but is dramatically reduced by systemic neutralization of IL-8 via intravenously administered anti-IL-8 antibodies. These data show that IL-8 plays an important role in the recruitment of PMNs during meningitis and that the physiological activity of IL-8 during this process appears to be on the blood site of the microvascular endothelium rather than on the parenchymal site. Thus, inhibition of IL-8 provides a possible therapeutic target for adjunct treatment of bacterial meningitis [41, 42]. The idea of an innate immune response in the brain during systemic inflammation is strongly supported by MCP-1 expression within the endothelium of the cerebral vessels, the circumventricular organs, the choroid plexuses, and in a few macrophages and microglial cells in response to systemically administered proinflammatory cytokines [43]. Intraperitoneal administration of anti-MCP-1 antibodies results in significant reduction of macrophages in the CSF and administration of anti-MIP-2 or anti-MIP-1a significantly decreases PMN infiltration during experimental bacterial meningitis [44]. At this point, it is appropriate to ask for the structural avenues which invading leukocytes follow during the process of immigration. Postcapillary venules represent the segment of the microvasculature most vulnerable to inflammatory processes. In experimental studies using transmission and scanning electron microscopy, it was found that PMNs migrate through the endothelium transcellularly. This initial process of extravasation includes: rolling and sticking of PMNs at the endothelium, adherence at the parajunctional endothelial domains, subsequent direct penetration of the


Fig. 24.1 Transmission electron microscopic studies show a direct extravasation of PMNs (Le) through the endothelium (En) in the initial phase of the inflammatory process (< 2 h). Note that the interendothelial junction (J) is not affected by the migrating leukocyte (Le). Bar = 1.5 lm.

endothelium or activation of the endothelial cells and engulfment of the PMNs by the endothelium, transendothelial passage, and subsequently penetration of the PMNs through the subendothelial basement membrane [45–47]. Morphological evidence for the involvement of interendothelial tight junctions in the initial process of PMNs extravasation through the inflamed BBB could not be obtained in any case (Fig. 24.1). A similar transendothelial extravasation is found for mononuclear cells under acute inflammation (Fig. 24.2) as well as under experimental autoimmune encephalomyelitis [48–50].

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Fig. 24.2 Set of transmission electron microscopic figures showing a sequence of serial sections of one mononuclear cell (Mo) in the initial phase of inflammation. Note that the emigrating cell is engulfed (arrows) by the endothelium (En). The interendothelial junctions (J) are not affected by the emigrating mononuclear cell. Bar = 1.0 lm.


24.3.3 Cytokines

Cytokines are key regulators of the immune response. Proinflammatory cytokines include TNF-a, IL-1b, -2, -6, -8, and interferon-gamma (INF-c).These cytokines are released early in the inflammatory process by endothelial and perivascular cells like macrophages and microglia [51, 52]. Cytokines influence the integrity of the BBB [53]. Increased CSF concentrations of TNF-a, IL-1b, IL-6, and IL-8 are established markers to discriminate acute bacterial meningitis from aseptic (viral) meningitis [54, 55]. In view of acute meningitis it is of interest that S. suis is able to stimulate the production of IL-6, IL-8, and MCP-1 by human brain microvascular endothelial cells but not human umbilical vein endothelial cells, in a time- and concentration-dependent manner [56]. Experimental interventions aimed at suppression of the effect of proinflammatory cytokines during bacterial meningitis may thus target different levels: 1. Direct blocking of the cytokine. TNF-a and IL-1b antibodies reduce meningeal inflammation in experimental meningitis when instilled into the CSF at the beginning of the infection [57]. Endogenous inhibitors of cytokines, such as recombinant IL-1 receptor antagonist or soluble TNF receptor, are effective against cytokine-induced meningitis but are not effective against LPS meningitis [58]. 2. Blocking of procytokine activation by proteolytic enzyme inhibition. Studies in a model of neonatal rat experimental bacterial meningitis show significantly down-regulated CSF levels of TNF-a by combined inhibition of matrix metalloproteinases and TNF-a-converting enzyme. In addition, the incidence of seizures, mortality, and neuronal injury is significantly decreased [59]. Similarly, treatment of rabbits and rats with experimental pneumococcal meningitis with the caspase-1 inhibitor z-VAD-fmk (benzyloxycarbonyl-Val-Ala-Aspfluoromethyl ketone) reduces CSF-PMN counts and IL-1b activation [60]. 3. Blocking of proinflammatory cytokines by anti-inflammatory cytokines. IL-10 and transforming growth factor beta (TGF-b) are considered prototypic antiinflammatory cytokines and potently inhibit the production of proinflammatory cytokines in vitro and in vivo [61, 62]. In experimental studies, therapeutic administration of IL-10 or TGF-b is consistently effective in modulating meningeal inflammation [63, 64]. However, in IL-10 gene-deficient mice, the absence of IL-10 is associated with higher proinflammatory cytokine and chemokine concentrations and a more pronounced PMN infiltrate, but antibacterial defense or survival of pneumococcal meningitis is not influenced [65]. 4. Regulation of cytokine production via interference with transcription factors, e.g. NF-jB. Various anti-inflammatory drugs including corticosteroids and anti-inflammatory cytokines such as IL-10 inhibit the NF-jB pathway. Pharmacological manipulation of NF-jB activation is investigated in experimental meningitis. Two agents have been tested, a NF-jB inhibitor, which interferes with IjB proteolysis, and another, which inhibits IjB phosphorylation. Both agents improved clinical status and reduced the BBB permeability and PMN extravasation into the CSF [66].

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24.4 Inflammatory Response in Acute Trauma

Traumatic injuries of the CNS initiate a complex cascade of events including an acute inflammatory response mediated by proinflammatory cytokines (see Chapter 11). IL-10 levels in the CSF and serum of patients with severe traumatic brain injury are related to IL-6, TNF-a, TGF-b1 and BBB function [67]. TNF-a levels have been associated with the neurological deficits in rodents subjected to experimental traumatic brain injury [68, 69]. In vivo experiments based on the neutralization of TNF-a after traumatic brain injury using common immunosuppressive agents or specific inhibitors showed a better neurological outcome and decreased brain oedema and less BBB dysfunction [68]. But recent studies on the role of cytokines after acute brain injury have yielded conflicting results with regards to their action contributing to repair mechanisms or exacerbating the pathophysiology of trauma [70]. Interestingly, posttraumatic mortality is significantly increased in TNF/lymphotoxin-deficient mice compared with wild-type animals, suggesting a protective effect of these cytokines. Moreover, neither the degree of BBB dysfunction nor the number of infiltrating PMNs in the injured hemisphere is different between wild-type and cytokine-deficient mice [71]. Neuropeptides like substance P and calcitonin gene-related peptide (CGRP) increase microvascular permeability, leading to oedema formation and neurogenic inflammation. Recent studies applied a capsaicin-induced neuropeptide depletion to examine the role of neurogenic inflammation following traumatic brain injury. Neuropeptide depletion yielded a significant reduction in posttraumatic oedema formation, BBB permeability, and both motor and cognitive deficits [72]. Traumatic injury is significantly greater in the spinal cord than in the cerebral cortex, as indicated by the number of PMNs recruited to the lesion site, which is significantly higher in the spinal cord as compared to the brain. Also, the area of BBB breakdown with concurrently occurring long-lasting vascular damage is substantially larger in the spinal cord [73]. A novel therapeutic peptide derived from apolipoprotein E (apoE) reduces brain inflammation and improves outcome after closed head injury [74]. Interestingly, apoE4, which was initially identified as a susceptibility gene for the development of Alzheimer’s disease, seems also to be associated with poor outcome after acute brain injury. One mechanism by which apoE may influence neurological outcome is by down-regulating the neuroinflammatory response. ApoE has been demonstrated to reduce glial activation and the CNS inflammatory response in vitro and in vivo in an isoform-specific action [75, 76], with the apoE4 isoform being less effective than apoE3 at down-regulating proinflammatory cytokines [75, 77].

24.5 Inflammatory Response in Alzheimer’s Disease

24.5 Inflammatory Response in Alzheimer’s Disease

Alzheimer’s disease (AD) is a chronic neurodegenerative condition that affects approximately 10% of individuals over 65 years. Five percent of this group of patients suffers from severe dementia. Memory decline as well as the inability to process new information are the most prominent clinical signs. Imaging techniques revealed disturbances in cerebral blood flow and glucose metabolism (see Chapter 25). A deficiency in the neurotransmitter acetylcholine has been suggested to be involved in AD, since cortical acetylcholine synthesis is markedly diminished in Alzheimer’s patients. Other neurotransmitters which may be involved in the course of the disease are dopamine, c-aminobutyric acid, vasoactive intestinal peptide, and glutamate. The pathology of AD is characterized by the extracellular deposition of a particular form of b-amyloid protein, derived from the larger precursor protein (APP). This amyloid peptide induces the release of the so-called b-peptide or the A4 peptide (A4P), which becomes stacked in the form of a b-pleated sheet structure with a high degree of intermolecular hydrogen bonding. Usually, the amyloid peptide precursor with an apparent molecular mass of 112 kDa is found in the brain. During AD, the A4P is processed abnormally into a 43-amino acid amyloiditic peptide that arises from near the C-terminus of the A4P. The pathology of an AD brain reveals granulovacuolar degeneration of neurons within the region of the hippocampus. Furthermore, neurofibrillary tangles are detected which are intraneuronal accumulations of dense non-membrane-bound fibrillary material forming paired helices. Other lesions are composed of amyloid, predominantly associated with microglial cells [78, 79]. Inflammatory reactions, surrounding the cerebral microvasculature, are frequently observed during AD. The number of perivascular macrophages increases and hypertrophy of astrocytes and microglia is observed in brain sections of AD patients [79]. IL-1 is found in the CNS of patients suffering from AD and its expression is thought to be an early event in the onset of AD. IL-1 affects the synthesis of b-amyloid precursor protein and the subsequent deposition of b-amyloid [80]. Increased levels of IL-6 and MCP-1 in the plasma and CSF of AD patients may also be good candidates as biomarkers for monitoring the inflammatory process in AD [81]. A stringent sign that the brain microvasculature is involved in the generation of AD pathophysiology is the accumulation of ICAM-1 in endothelial cells as well as in senile plaques [82], indicating that infiltration of lymphocytes into the brain tissue accompanies the inflammatory reactions during AD. CSF studies showed an increased level of soluble ICAM-1, but not soluble E-selectin (s-ELAM-1) in AD, suggesting a more neural than endothelial s-ICAM origin in patients with AD [83]. Albumin concentrations are also enhanced in the CSF of Alzheimer’s patients at the early onset of AD, apparently resulting from an increased permeability in the BBB [84]. Two-dimensional gel electrophoresis showed the presence of a protein, haptoglobulin, with a molecular mass of 13.5 kDa in the CSF of AD

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patients. The presence of this protein was suggested to be the result of an increased penetration of haptoglobin across an impaired BBB [85]. AD patients have a diminished density of mitochondria and feature incomplete interendothelial junctions in the cerebral endothelial cells, suggesting leakiness of the vessels [86]. The diminished cerebral metabolic rate observed during AD can be partly explained by a decreased density of glucose transporters (GLUT-1) in the brain capillaries [87, 88]. Changes in intracellular signal transduction in cerebral microvessels of AD patients, in comparison with age-matched controls, have also been observed. Protein kinase C activity is strongly diminished in the brain microvessels, which leads to altered protein phosphorylation. Protein kinase C activity during AD seems to be important in the inhibition of the processing of b-amyloid precursor protein into soluble Ab protein. Intracellular signaling in the cerebrovasculature may be one of the pathophysiological targets in AD; and the balance of various secondary messenger systems may be disturbed [89]. In AD transgenic mice, the therapeutic effects of the PKC activator bryostatin-1 included improvement of behavioral outcomes [90]. Perivascular inflammatory reactions during AD are likely to cause additional changes in the function of the BBB. Recent studies showed that the BBB is disrupted in brains of AD patients. In addition, cultures of endothelial cells derived from brain capillaries of AD patients revealed the production of the b-amyloid precursor protein. Apolipoprotein E4, which is considered a risk factor for the development of AD, is also produced by cerebral endothelial cells [91]. Brain microvessels from AD patients express high levels of inflammatory proteins, suggesting that the brain microcirculation could be a source of neurotoxic factors in AD [92]. Taking together, all indicated studies provide substantial evidence that the function of the BBB is impaired in AD patients. Disruption of the BBB can lead to the entry of neurotoxic environmental factors into the brain or circulating amyloid. One hypothesis is that BBB opening may be the initial insult that causes AD, although more research needs to be conducted to substantiate this hypothesis.

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Acta Neuropathol. 91, 608. 83 M. Rentzos, M. Michalopoulou, C. Nikolaou, C. Cambouri, A. Rombos,

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A. Dimitrakopoulos, E. Kapaki, D. Vassilopoulos 2004, J. Geriatr. Psychiatry Neurol. 17, 225. P. Mecocci, L. Parnetti, G. P. Reboldi, C. Santucci, A. Gaiti, C. Ferri, I. Gernini, M. Romagnoli, D. Cadini, U. Senin 1991, Acta Neurol. Scand. 84, 210. K. M. Mattila, T. Pirtilä, K. Blennow, A. Wallin, M. Vitanen, H. Frey 1994, Acta Neurol. Scand. 89, 192. P. A. Stewart, K. Hayakawa, M. A. Akers, H. V. Vinters 1992, Lab. Invest. 67, 734. S. I. Harik 1992, Can. J. Physiol. Pharmacol. 20[suppl], S113. I. A. Simpson, K. R. Chundu, T. Davies-Hill, W. G. Honer, P. Davies 1994, Ann. Neurol. 35, 546. P. Grammas, P. Moore, T. Botchelet, O. Hanson-Painton, D. R. Cooper, M. J. Ball, A. Roher 1995, Neurobiol. Aging 4, 563. R. Etcheberrigaray, M. Tan, I. Dewachter, C. Kuiperi, I. van der Auwera, S. Wera, L. Qiao, B. Bank, T. J. Nelson, A. P. Kozikowski, F. van Leuven, D. L. Alkon 2004, Proc. Natl Acad. Sci. USA 101, 11141. J. M. Wells, A. Amaratunga, D. C. McKenna, C. R. Abraham, R. E. Fine 1995, Int. J. Exp. Clin. Invest. 2, 229. P. Grammas, T. Ottman, U. Reichmann-Philipp, J. Larabee, P. H. Weigel 2004, J. Alzheimer Dis. 6, 275.

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25 Stroke and the Blood-Brain Interface Marilyn J. Cipolla

25.1 Introduction

Ischemic injury of the brain is one of the most common pathophysiologic processes affecting more than 750,000 people per year in the United States in the form of stroke [1]. Brain edema with a subsequent rise in intracranial pressure is the most dangerous complication of stroke [2], often occurring during the reperfusion period after recanalization of an occluded artery (i.e., reperfusion injury) [3]. Increased cerebrovascular permeability is considered the most important factor for development of cerebral edema and is determined by the cerebral endothelial cells (EC) that form the blood-brain barrier (BBB) [4, 5]. The morphologic features of cerebral ECs that prevent the extravasation of large and small solutes are the presence of tight junctions that reduce paracellular transport and the low rate of pinocytotic vesicle formation that limits transcellular transport [5–7]. Recently, it has become evident that these processes are regulated by factors that influence both EC structure and function, including hemodynamic factors such as flow and pressure [8–10], the production of autocoids and second messengers such as nitric oxide (NO), superoxide anion (O–2), and protein kinase C (PKC) [11–13], and the organizational state of the EC actin cytoskeleton [14–16]. Many of these same factors that regulate permeability under normal conditions are known to be altered during ischemia and/or reperfusion, causing enhanced vascular permeability and edema formation. The purpose of this chapter is to present our current understanding of alterations in cerebral EC structure and function due to ischemia and reperfusion (I/R) that promote BBB disruption and cerebral edema formation.


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25.2 Brain Edema Formation During Stroke

The development of brain edema is one of the most detrimental consequences of stroke and is a significant determinant of stroke outcome [17, 18]. Studies have demonstrated that edema, increased intracranial pressure, and brain herniation are the major determinants of not just patient outcome, but survival as well [19]. Secondary, progressive ischemia due to vascular compression and elevated intracranial pressure are also thought to contribute to the pathogenesis of stroke, demonstrating the significant impact of brain edema [20]. Furthermore, while it is difficult to separate evolving edema due to ischemia from the damage that occurs during the reperfusion phase, there have been numerous cases of severe edema thought to be due to postischemic reperfusion [21–23], indicating that damage is not restricted to the ischemic periods, but occurs during reperfusion as well (i.e., reperfusion injury).

25.2.1 Cytotoxic Versus Vasogenic Edema

Klatzo first characterized brain edema as cytotoxic versus vasogenic, depending on whether or not the BBB is disrupted [24]. In the simplest terms, cytotoxic edema occurs when brain cells swell at the expense of the extracellular space while the BBB remains intact. In vasogenic edema, cerebrovascular permeability is increased due to BBB disruption that allows an influx of plasma constituents and expansion of the extracellular space. During stroke, features of both cytotoxic edema and vasogenic edema occur simultaneously (Fig. 25.1, see pp. 622/ 623). Ischemia causes cells to lose their normal ionic gradients due to diminished ATP and loss of energy to drive ionic pumps [25]. Since sodium entry exceeds potassium loss, a net increase in intracellular ions occurs [26–28]. Consequently, water enters the cell due to abnormal osmotic gradients and cytotoxic edema ensues [25–30]. Theoretically, because the exchange of water and ions is between the cell and the extracellular space (i.e., no BBB disruption), there is no net increase in brain water content and therefore this is not true cerebral edema. However, the ionic gradient will eventually be sufficient to affect the cerebral endothelium, causing a net increase in water from the plasma [29]. This enhanced hydraulic conductivity increases the bulk flow of ions into brain tissue from the plasma due to hydrostatic pressure. A close correlation exists between the net increase in brain cations and edema formation. In fact, the rate of sodium flux across the endothelium may control the rate of edema formation [26, 27, 29, 30]. During continuous ischemia, the BBB becomes disrupted and ions enter the brain more rapidly, causing significant brain edema and the passage of potentially toxic substances into brain tissue [31]. Under normal conditions, the barrier properties of the cerebral endothelium that forms the BBB has low hydraulic conductivity that prevents bulk flow of

25.3 Role of Astrocytes in Mediating Edema During Ischemia

water, ions and proteins into the brain from hydrostatic forces such as blood pressure [32]. When the BBB is disrupted (e.g., during ischemia), hydrostatic forces become significant enough that the rate of protein entry into the brain is directly related to the pressure gradient between the blood and the brain. However, while the passage of protein from the plasma into the brain is a measure of BBB disruption, it does not significantly contribute to edema formation. Albumin and other proteins passing into the brain have been used as a measure of the BBB disruption that leads to edema formation. However, the concentration of a large protein is several orders of magnitude smaller than that of ions. Therefore, the increase in osmolality that occurs when albumin enters the brain is small compared to that of ions [32]. Albumin and protein entry into the brain may be an indicator of BBB disruption, but its contribution to edema formation is small compared to that of ions. It is important to note that cell swelling associated with cytotoxic edema may not be entirely detrimental. Under certain conditions, the cell volume increase may be compensatory or even protective. Precapillary astrocyte endfeet are the first cellular elements to swell during ischemia [33], a process thought to normalize the composition of the extracellular environment for normal neuronal activity [34]. Glial cells can also inactivate neurotransmitters [35], take up excess potassium ions produced during neuronal activity [36], and scavenge reactive oxygen species [37].


While it is the structural properties of the cerebral endothelium that make up the BBB (e.g., complex tight junctions, low rate of pinocytosis, lack of fenestrations), surrounding cells within the brain parenchyma are known to contribute to and induce BBB properties, most notably astrocytes. An early view of the BBB was that astrocytic endfeet, which are anatomically in close apposition to the cerebral endothelium and brain parenchymal vessels, provided a physical barrier and thus contributed to BBB properties. However, studies by Reese and Karnovsky showed that the site of barrier function was at the level of the cerebral endothelium and not the astrocytes [38, 39]. A role for astrocytes in inducing the BBB properties of the cerebral endothelium, as opposed to being a physical barrier, emerged around the same time [40]. Subsequent in vitro cell culture studies confirmed the important role of glial cells in inducing BBB phenotype, including upregulation of tight junction proteins [41, 42]. There is also considerable evidence that the cerebral endothelium influences and signals astrocytes. For example, the water channel aquaporin 4 (AQP4) is primarily expressed only in astrocytic endfeet surrounding parenchymal vessels, but not in astrocytes just interacting with neurons [43]. In support of this concept, AQP4 has been shown to upregulate in astrocytic endfeet when cocultured with endothelium [44] (see Section 25.3.1). Therefore, the maintenance of BBB properties and function likely depends on cross-talk between the endothe-

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Fig. 25.1 Development of cerebral edema formation during ischemia. (a) Normal blood-brain barrier properties. Astrocytic foot processes closely appose the cerebral endothelium and contain aquaporin water channels and ionic pumps (Na-K-ATPase) that control the intracellular ionic environment and cell swelling. The cerebral endothelium, due to its structure, has low hydraulic conductivity that limits bulk flow of water from hydrostatic forces. Tight junction proteins are linked to the actin cytoskeleton and allow the regulation of paracellular flux by mediators that affect the contractile state of the cell and/or actin

cytoskeletal dynamics. Pinocytotic vesicles are present in low number and contribute to normal brain homeostasis. (b) Initial stages of ischemia. Ischemia causes cells to lose their normal ionic gradients due to the loss of energy to drive ionic pumps (Na-K-ATPase). Because sodium influx exceeds potassium loss, there is a net increase in intracellular sodium that causes water to enter the cell due to osmotic forces. The astrocytes swell (cytotoxic edema), but since cell swelling is at the expense of the extracellular space, this is not true edema formation.


(c) Later stages of ischemia. Eventually the ionic gradient becomes sufficient to pull water across the cerebral endothelium from the plasma, increasing hydraulic conductivity. This is true cerebral edema formation, because of the net increase in brain water content. (d) Prolonged ischemia. During continuous ischemia, the blood-brain barrier becomes

disrupted and ions enter more rapidly, causing significant cerebral edema. At this stage, tight junctions are disrupted and there is an increase in vesicular transport that also increases extravasation of proteins and other toxic substances from the blood into the brain parenchyma. Aquaporin water channels become upregulated in an attempt to normalize intracellular water content.

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lium and astrocytes. Because of the interdependence of endothelium and glia, disruption of any of these signals during ischemia can result in disruption of function. Pial vessels also have BBB properties, even though they are not directly in contact with astrocytes [45, 46]. The BBB extends throughout the brain with the exception of the circumventricular organs. The fact that pial vessels show BBB properties suggests that soluble factors from the glia limitans or subarachnoid CSF may be the source [46]. Butt et al. measured transendothelial electrical resistance (TEER) in pial vessels in situ and found that they have an electrical resistance of around 1800 X cm–2, a number that is somewhat less than the microcirculation, but considerably greater than cell culture models of BBB, even when cocultured with astrocytes. During ischemia, astrocytes are known to be protective of both neurons and endothelium and maintain barrier properties. For example, Fischer et al. [47] showed that hypoxia-induced hyperpermeability of cultured porcine brain-derived microvascular endothelial cells was significantly attenuated when either cocultured with astrocytes or in glial-conditioned media. This effect was shown to be due to prevention of hypoxia-induced restructuring of the tight junction protein zona occludens-1 (ZO-1). Other studies have shown that astrocytes confer a protective role on the BBB during hypoxia and reoxygenation. Cerebral microvascular endothelial cells cocultured with astrocytes have reduced decrease in barrier properties, due to hypoxia/reoxygenation compared to endothelial cells alone [48]. The presence of astrocytes has also been shown to prevent upregulation of hypoxia-induced vascular endothelial growth factor (VEGF) expression in endothelial cells, a known contributor to increased vascular permeability during stroke [49]. Another role of astrocytes that may be important in BBB function during ischemia is providing a secondary barrier to endothelial disruption by the ability to swell and maintain ionic homeostasis. This cytotoxic edema, likely mediated by aquaporins [50, 51], can influence ionic composition of the extracellular space and metabolize unwanted protein and neurotransmitter that passes from the blood into the brain [33, 34]. The protective effect of astrocytes is limited though. Eventually, with the BBB disrupted, hydrostatic pressure becomes a significant influence, and together with this cytotoxic edema, intracranial pressure increases and causes neurologic complications. However, this protective role of astrocytes makes them a potential therapeutic target for ischemia-induced edema formation.

25.3.1 Aquaporins and Cerebral Edema During Ischemia

An important factor in determining cytotoxic swelling of astrocytes is the aquaporins (AQPs). The AQPs are a family of channel-forming transmembrane proteins that facilitate the movement of water, glycerol, and other solutes across the plasma membrane of cells [52–55]. AQP4, the predominant aquaporin in the brain, has been localized in the astrocytic endfeet surrounding blood vessels,


but has also recently been found in the vascular endothelium [56–59]. One role of astrocytic AQP4 may be in the actual regulation of water transport across the BBB, mainly because of the highly polarized localization of AQP4 in the perivascular endfeet of astrocytes (see Chapter 9) [54, 55, 60]. This concept is supported by numerous studies that have shown increased AQP4 expression during conditions that cause vasogenic brain edema, including brain tumors, focal ischemia, and brain injury [60–64]. Whether the increase in AQP4 expression during ischemia is beneficial or detrimental is still unclear. AQP4 in astrocytes is thought to contribute to BBB properties by taking up excess water brought into the brain by disruption of the BBB. However, excessive AQP4 may be detrimental and promote increased intracranial pressure, due to excessive cytotoxic edema formation. For example, knockout mice in which deletion of a-syntrophin, a membrane protein anchoring AQP4 into the perivascular astrocytic endfeet, resulted in the loss of AQP4 at these membranes [59]. Following middle cerebral artery (MCA) occlusion and reperfusion, a-syntrophin knockout mice showed decreased levels of hemispheric enlargement and brain edema compared to wild-type mice, suggesting that the presence of perivascular AQP4 enhanced brain edema formation [59]. Another study using AQP4 knockout mice found that, following vasogenic edema and BBB disruption from MCA occlusion and focal cerebral ischemia, AQP4+/+ mice had a higher mortality rate and greater neurological complications than AQP–/– mice. In addition, the AQP–/– mice had significantly less brain edema, as measured by the increase in the size of the cerebral hemisphere, suggesting AQP4 promotes edema formation [63]. In a model of permanent focal brain ischemia, the pattern of AQP4 expression followed both the formation and resolution of edema formation, suggesting that AQP4 has a role in post-ischemic edema formation and possibly resolution of edema [62]. Investigating specific changes in AQP4 during ischemia and reperfusion may not provide a complete picture of the pathologic consequences of edema formation. An important consideration of the impact of edema formation is edema resolution. Because the cerebral endothelium does not contain fenestrations and the brain lacks a lymphatic system, edema resolution is through the choroid plexus epithelium and cerebral spinal fluid (CSF) that turns over every 4 h [65]. AQP1 is localized to the choroid plexus epithelium and is thought to be involved in both the secretion and reabsorption of CSF [66]. Due to its role in CSF reabsorption, AQP1 may be an important factor in edema resolution under ischemic conditions. However, a role for AQP1 in ischemia has yet to be determined. In addition, at least six AQPs have been found in the rodent brain at various locations or expressed in brain-derived cells [50]. The role of these AQPs in postischemic edema formation and resolution is also unclear, but likely to have a significant impact due to their water fluxing properties.

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25.4 Cellular Regulation of Cerebrovascular Permeability 25.4.1 Role of Actin

Morphologically, brain ECs are more similar to epithelial cells than to ECs in peripheral blood vessels in that there is limited molecular transport due to a low rate of fluid-phase endocytosis (that limits transcellular flux) and coupling by high electrical resistance tight junctions (that limits paracellular flux) [3–5]. Significant steps have been made in understanding the molecular structure of tight junctions in brain ECs that affords these features. It is becoming increasingly apparent that tight junctions are not passive structures, but can be rapidly modulated by signaling pathways that affect the structure of the tight junction and the organization of the EC actin cytoskeleton [4, 5, 14, 67, 68]. At least ten proteins have been localized to the tight junction, some of which are involved in signal transduction processes that regulate permeability and are directly attached to the actin cytoskeleton [67, 68]. For example, within the tight junction, actin filaments bind to the zona occludens, forming a direct connection from the paracellular space through the membrane to the cytoplasm [4, 5, 69], providing a mechanism of regulation by factors that influence the actin cytoskeleton (see Chapters 1 and 4). The contractile activity of EC actin stress fibers within the cytoplasm is of central importance to regulating tight junction permeability. Agonists that promote relaxation of stress fibers (e.g., cyclic-AMP) decrease permeability by cell spreading that strengthens cell-cell contact and reduces paracellular transport [15]. Alternatively, agonists that promote stress fiber contraction (e.g., PKC, VEGF) promote increased permeability by causing cell rounding that decreases cell-cell contact [70]. This hypothesis is supported by several studies that have demonstrated that inhibition of myosin light-chain (MLC) phosphorylation, which inhibits actin stress fiber contraction, decreases agonist-induced permeability [71– 73]. Stress fiber activity as an underlying mechanism of paracellular transport is an important consideration for the numerous mediators of cerebral edema formation that are produced by the injured brain, e.g., histamine, bradykinin, arachidonic acid, etc. (see Section 25.6). Further evidence for the involvement of EC actin in maintaining barrier function is demonstrated by numerous studies that have shown that depolymerization of actin filaments with cytochalasin increased permeability in both epithelial and ECs and that stabilization of the cytoskeleton with either phalloidin or jasplakinolide (compounds that specifically bind to and stabilize F-actin) prevented the increased permeability [14, 16, 74–77]. In addition to affecting paracellular flux, the polymerization state of actin in ECs has also been shown to affect pinocytotic vesicle formation and transcellular flux. Rats infused with cytochalasin B, a cell-permeable compound that disrupts actin filaments, had increased pinocytotic vesicle formation in cerebral ECs and enhanced transcellular

25.4 Cellular Regulation of Cerebrovascular Permeability

transport of electron-dense horseradish peroxidase (HRP) into the brain cortex [16]. However, these conditions likely mimic endothelial cell damage and possibly necrosis that occurs after longer periods of cerebral ischemia or due to severe reperfusion injury.

25.4.2 Ischemia and Hypoxia Effects on EC Actin and Permeability

Hypoxia has been shown to affect the state of actin in ECs and influence permeability in vitro. For example, in cultured brain endothelial cells exposed to hypoxia, a dramatic increase in permeability to sucrose was noted that correlated with increased filamentous (F-)actin and stress fiber formation [13, 78]. Other studies have found that hypoxia with subsequent reoxygenation increased F-actin by 41% in cultured ECs and changed the morphology of ECs to be more spindle-shaped [74]. Alternative to these findings, several studies have suggested that EC damage, including during hypoxia/reoxygenation, disrupts F-actin and leads to increased permeability [13, 79], suggesting that the duration of ischemia or hypoxia impacts the cellular processes that influence permeability. In cultured brain ECs, long periods of hypoxia were associated with a marked increase in permeability that was attributed to ATP depletion that caused depolymerization of EC actin filaments [13]. It therefore appears that there is a gradient of damage to the EC actin cytoskeleton, such that short periods of hypoxia increase stress fiber formation and enhance paracellular permeability, whereas longer periods cause depolymerization and overall EC permeability that may also include transcellular transport. The underlying cellular mechanisms by which I/R affects the EC actin cytoskeleton are not clear, but may relate to ATP depletion during ischemia [13] and/or the production of autocoids (NO, O–2) during reperfusion (see Section 25.5) [13, 80]. In a cell culture system, the role of EC actin in reperfusion injury was studied by exposure of cerebral microvascular endothelial cells to posthypoxic reoxygenation. Mark and Davis found that hypoxia induced a 2.6-fold increase in paracellular permeability to sucrose that was reduced by 58% during reoxygenation [78], suggesting that reperfusion may actually decrease permeability. While these in vitro studies importantly demonstrate that hypoxia has cellular effects on the cerebral endothelium that promotes changes in permeability, the true impact in the brain where there are complex interactions between hemodynamic factors and chemical mediators are less clear.

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25.5 Reperfusion Injury

The mechanisms by which ischemia promotes brain tissue damage and cerebral edema are likely different from those during reperfusion. During ischemia, there is reduced oxygen delivery that diminishes ATP and causes metabolic disturbances, whereas during reperfusion there is repletion of ATP and release of damaging O–2 [3]. Increased permeability of cerebral vessels after brain ischemia is thought to involve the opening of EC tight junctions and increased vesicular transport, together with F-actin and tight junction protein rearrangements that promote paracellular opening. During reperfusion, the release of various substances, including O–2, can affect EC structure and function to increase permeability, as well as promote hyperemia that has been shown to cause vasogenic edema [81–83] (see Section 25.9). Therefore, there is clearly a differential effect of ischemia versus reperfusion on vascular permeability and BBB properties.

25.5.1 Nitric Oxide and Other Reactive Oxygen Species as Mediators of EC Permeability During Ischemia and Reperfusion

Nitric oxide (NO) is a ubiquitous intercellular signaling molecule synthesized in several cell types, including neurons, endothelium, and macrophages, from the amino acid l-arginine by the enzyme nitric oxide synthetase (NOS) [84]. Large amounts of NO are produced within minutes after MCA occlusion within the cortex and vascular endothelium [85, 86]. The initial increase in NO during ischemia is thought to be neuroprotective by improving cerebral blood flow (CBF). However, the sustained increase in NOS activity is thought to contribute to ischemic injury [87, 88]. The detrimental effects of NO production during ischemia are demonstrated in numerous studies showing reduced infarct size in animals genetically deficient in the neuronal isoform of NOS, and by inhibiting NOS activity during reperfusion [87–90]. One major mechanism of injury associated with NO production in vivo is due to its reaction with superoxide anion in an irreversible, diffusion-limited reaction to form peroxynitrite (ONOO–) [84]. Reactive oxygen species (ROS), such as O–2 and hydrogen peroxide (H2O2) are produced in significant amounts during the reperfusion phase following ischemia and have been implicated in reperfusion injury by their ability to incur oxidative damage to lipids, proteins and nucleic acids, as well as their ability to interact with other compounds such as NO to form more reactive adducts [84, 91–95]. The production of ONOO– during reperfusion seems likely since large amounts of NO produced during ischemia are available to combine with O–2 generated during reperfusion. Several studies have demonstrated ONOO– production during ischemia and reperfusion which has led to the hypothesis that it is a mediator of reperfusion injury [95–97]. As an anion, peroxynitrite

25.5 Reperfusion Injury

is remarkably stable and is particularly effective at oxidizing electron-rich groups such as thiols, iron/sulfur centers and zinc fingers [84]. Increased vascular permeability in response to increased NO, O–2, and ONOO– production has been demonstrated in numerous studies using cultured cells [79, 98–101], many of which noted altered EC F-actin and damage as well [100]. For example, significant amounts of O–2 and H2O2 were measured in cultured cells within minutes of reoxygenation after being exposed to hypoxia that was associated with EC damage [99]. This effect was blocked by allopurinol, a compound that inactivates O–2. Similarly, Lum et al. found that hypoxia/reoxygenation increased permeability of ECs to albumin two- to five-fold [79]. The increased permeability was associated with significant F-actin reorganization in ECs. Treatment of the cells with superoxide dismutase (SOD) prior to hypoxia prevented the increase in O–2 and 50% of the increase in permeability. Other in vitro studies have shown that damage to cerebral ECs due to posthypoxic reoxygenation was attenuated by the presence on both SOD and L-NAME, suggesting that ONOO– is a primary mediator of EC injury during hypoxia/reoxygenation [101]. In vivo studies have also demonstrated a role for ONOO– in mediating reperfusion-induced cerebral edema. Inhibition of NO with L-NAME after 3 h of ischemia reduced BBB disruption by 65% and improved stroke outcome in a mouse model of transient focal ischemia [102]. A similar study in rats found that NO inhibition with L-NAME reduced ischemic brain damage when given just before reperfusion after 2 h of ischemia that was associated with diminished immunoreactivity for nitrosotyrosine, a marker for ONOO–, suggesting that ONOO– formation contributes to reperfusion-induced cerebral edema [95].

25.5.2 Thresholds of Injury

It is well known that the depth and duration of ischemia affect the magnitude of stroke outcome, giving rise to the threshold concept of cerebral ischemia [103–105]. Flow and time thresholds for neuronal injury and edema formation have been established in humans and many experimental animal models. For example, one study in baboons found that when CBF fell below 35–40% of normal, the time threshold for edema formation was 30 min [105]. The cellular mechanisms that underlie vascular permeability are also likely to be affected by the duration of ischemia and reperfusion. For example, in cultured brain ECs, hypoxia was shown to affect F-actin in a threshold-dependent manner [13]. Short periods of hypoxia were shown to cause increased F-actin, but longer periods caused actin depolymerization that correlated with apoptosis and ATP depletion. This suggests there may be a threshold duration of ischemia for actin cytoskeletal changes that underlies vascular permeability. Understanding these thresholds is important when considering the window of opportunity for therapeutic intervention.

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25.6 Transcellular Transport as a Mechanism of BBB Disruption During Ischemia

The passage of blood-borne substances into the brain parenchyma via vesicular or transcellular transport is considerably less studied during ischemia and reperfusion, but is by no means a less important route of passage than the paracellular route. The paucity of information regarding transcellular transport as a mechanism of enhanced cerebrovascular permeability, compared to that of the paracellular (tight junction) opening, is likely due to the fact that cell culture studies do not easily lend themselves to measuring transcellular transport. A great deal of information regarding how tight junctions are structured and affected by conditions such as hypoxia and reoxygenation have been gained from cell culture studies that measure transendothelial electrical resistance (TEER) as an indicator of barrier disruption. Because TEER is a measure of ionic flux, this information is limited to the paracellular route. A few studies have used cultured endothelium to investigate transcellular uptake of protein tracers during hypoxia, which is distinguished from the transcellular route by inhibition at low temperature [106]. Evidence for a role of pinocytosis in vascular permeability is largely circumstantial, but has suggested that this mode of transport is important for enhancing the permeability of the BBB under pathologic conditions. Under normal conditions, the majority of pinocytosis is from apical to basolateral [9, 107], which argues against bidirectional trafficking through the endothelium and supports the concept of a BBB. Electron microscopy studies have shown that the number of apical vesicles that are apparent in endothelium correlate with how permeable the vessel is. Vesicles are found in large numbers in vessels known to have high permeability (e.g., skeletal muscle arterioles) and low numbers in vessels with low permeability (e.g., cerebral) [108]. In cerebral endothelium that has BBB properties, vesicle formation is low under normal conditions, but increases substantially under experimental and pathologic conditions in which barrier permeability is enhanced [9, 109, 110], suggesting that this mode of transport contributes to enhanced permeability and BBB disruption. There is evidence that transcellular transport from blood to brain is enhanced during ischemia and reperfusion [9, 106, 111–114]. Using a coculture model of brain capillary endothelial cells and astrocytes, Plateel demonstrated hypoxia-induced permeability and uptake of albumin was temperature-sensitive, suggesting that enhanced vesicular transport is a means of albumin accumulation during hypoxia [106]. However, in a model of focal ischemia without reperfusion, transfer constants for sucrose and inulin were measured and showed that convective, fluid-phase endocytosis transport was not increased [115]. The difference in these studies may be due to measuring vesicular transport (pinocytosis) versus fluid-phase endocytosis (membrane turnover). The polarized nature of the cerebral endothelium not only contributes to barrier properties, but may also provide a mechanism of protection. Enzymatic substances on the abluminal or basolateral membrane inactivate neurotransmitters

25.7 Mediators of EC Permeability During Ischemia

and remove toxins from the brain into the bloodstream [116, 117]. Studies from our laboratory have shown that ischemia and reperfusion increase basolateral pinocytosis in cerebral endothelium, suggesting an efflux mechanism is invoked to rid the brain of toxic substances that may accumulate during ischemia, or to reduce intracranial pressure that has been elevated during edema formation, since vesicular transport has been shown to be pressure-induced [9]. There are several mechanisms by which transcellular transport can increase apical to basolateral and promote edema formation. First, mediators of cerebral edema such as bradykinin and histamine that are released during ischemia have been shown to enhance permeability by vesicular (receptor-mediated) transport [118] (see Section 25.7). Second, prolonged ischemia causes actin depolymerization that could also increase pinocytosis. Nag et al. showed a significant increase in pinocytosis in the cerebral cortex of anesthetized rats infused with cytochalasin B, a cell-permeable compound that causes depolymerization of actin [16]. A role for actin in mediating transcellular transport is likely given that the endocytotic machinery is directly linked to the actin cytoskeleton [119]. Last, changes in cerebral hemodynamics due to postischemic reperfusion likely contribute to an increase in apical pinocytosis, since pressure has been shown to increase pinocytotic vesicle formation and the passage of HRP through the cerebral endothelium [9]. It is important to note that many studies that show enhanced transcellular transport or pinocytosis during ischemia/reperfusion or hypoxia/reoxygenation have not always shown an increase in transport into the brain parenchyma. While this mode of transport likely contributes to enhanced vascular permeability, more evidence is needed to ascertain its specific role in cerebral edema formation.

25.7 Mediators of EC Permeability During Ischemia

The increase in BBB permeability and edema formation during stroke is also partly due to the production of autocoids by the injured brain tissue and endothelium. In general, a mediator has the properties that it can: (a) increase BBB permeability, (b) have the capacity to induce brain edema, (c) cause vasodilation that augments permeability due to increased surface area, and (d) increase its concentration in the brain during ischemia and/or reperfusion [118, 120]. These criteria provide a framework for which therapeutic interaction may influence cerebral edema formation. The following is a list of some of the known mediators of cerebral edema during ischemia and reperfusion. Some of these are inflammatory mediators and it is important to note that there is an inflammatory response associated with stroke that is thought to cause secondary injury to brain tissue following ischemia. Because inflammatory mediators of BBB permeability are nicely presented in Chapter 24, they will only be mentioned here in the context of how they are involved in BBB disruption and cerebral edema during ischemia.

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1. Bradykinin (BK) is an endogenous nonapeptide produced by activation of the kallikrein-kinin system that has been shown to increase in brain tissue after ischemia and cause brain edema [121, 122]. It has been shown to increase BBB permeability by both vesicular transport and paracellular mechanisms (i.e., decreased TEER) [123–125]. It is a potent vasorelaxant by endotheliumdependent vasodilation due to release of NO, free radicals, leukotrienes, and prostanoids [126–128]. Inhibition of the BK B2 receptor significantly reduces postischemic brain injury in both rat and mouse models [129, 130]. 2. Histamine (HA) is an inflammatory mediator that is increased in brain tissue after ischemia, likely due to release from mast cells [131, 132]. It causes significant brain edema formation and BBB permeability by both vesicular transport and paracellular tight junction opening (measured by TEER) [46, 125, 133, 134]. It dilates pial vessels by activation of H2 receptors on smooth muscle and H1 receptors on endothelium [118, 135, 136]. Histamine antagonists have been shown to reduce accumulation of water and sodium in brain tissue after bilateral common carotid artery occlusion in rats [137]. 3. Arachidonic acid (AA) is increased after focal ischemia and causes nonspecific opening of the BBB [138, 139]. Its vasomotor activity has been assessed in pial arterioles and found to both cause constriction and dilation [141, 142]. Application of glucocorticoids to decrease phospholipase A2 successfully decreased brain edema after ischemia [143, 144]. 4. Endothelin-1 (ET-1) increases in brain tissue and plasma after cerebral ischemia [145] and has been shown to increase BBB permeability [146, 147]. While ET-1 may not fit the category of mediator because it causes potent vasoconstriction that is thought to further promote ischemia instead of dilation [148, 149], its inhibition by ET-A receptor antagonists has been shown to be effective at decreasing postischemic brain edema and improving stroke outcome [150, 151]. 5. Vascular endothelial growth factor (VEGF) is a secreted mitogen whose expression is increased by hypoxia and ischemia [152–154]. It is a potent permeability factor that increases BBB permeability by both transcellular and paracellular mechanisms [155, 156]. It also produces potent vasorelaxation [157] and can therefore be considered a mediator of cerebral edema. Its role in stroke outcome is complex. Because of its mitogen activity, it can increase angiogenesis of cerebral microvessels after stroke and improve outcome, but its permeability effects preclude it from being an effective or beneficial treatment. However, due to the fact that ischemia-induced permeability increases in the acute stage (within minutes), while angiogenesis develops much later in the ischemic brain, Zhang et al. demonstrated that late (48 h) administration of recombinant VEGF to rats after ischemia significantly improved stroke outcome due to increased microvessel density [158].

25.8 Hyperglycemic Stroke

25.8 Hyperglycemic Stroke

Serum glucose concentration is a known determinant of stroke outcome [159– 163]. Diabetic patients have a significantly increased magnitude of stroke that has been attributed to hyperglycemia and not to other complicating factors associated with diabetes, such as atherosclerosis or microangiopathy [161, 164, 165]. In animal models, preexisting hyperglycemia has been shown to triple infarct size as a result of permanent MCA occlusion and to increase the incidence of fatal edema seven-fold during transient ischemia [166, 167]. Therefore, the pathologic processes that increase vascular permeability and promote brain edema during ischemia and reperfusion are severely aggravated by the presence of high glucose (see Chapter 26). Hyperglycemia has been shown to increase lactate production [168] and impair ionic hemostasis during ischemia, which is thought to contribute to brain tissue damage [169]. While these factors may have a role in enhancing edema formation during ischemia, the mechanisms of enhanced edema formation during reperfusion, when ATP levels are restored and production of autocoids and second messengers is pronounced, is likely very different.

25.8.1 Role of PKC Activity in Mediating Enhanced BBB Permeability During Hyperglycemic Stroke

There is extensive literature implicating a role for protein kinase C in regulating tight junction permeability [3, 4, 170–174]. Agonists that are known to increase vascular permeability both in vivo and in vitro, including bradykinin and thrombin, activate PKC through the phospholipase C (PLC)-diacylglycerol (DAG) pathway [172, 173]. This thrombin- and bradykinin-induced increase in permeability did not occur in ECs depleted of PKC [172–174], suggesting a central role for PKC activity in modulating permeability. In addition, specific inhibitors of PKC, including calphostin C and H-7, prevented agonist-induced permeability [172, 173, 175] in ECs; and several studies have shown that PKC activation by phorbol esters increases paracellular permeability [176]. The involvement of PKC in increasing permeability during hyperglycemia may relate to PKC-induced affects on EC actin. PKC activation phosphorylates cytoskeletal proteins, leading to F-actin rearrangements [177]. PKC has been shown to colocalize with ZO-1 at the tight junction and is thought to affect permeability through its ability to phosphorylate tight junction proteins such as ZO-1 [178], directly altering the structure of the tight junction and attachment to the cytoskeleton. In addition, PKC activation, through its ability to promote calcium/calmodulin-dependent actin-myosin interaction, is thought to increase permeability by causing EC contraction [174, 175, 179, 180] and increasing intercellular gap formation. This effect was demonstrated by one study which showed that PKC-induced permeability was prevented by the inhibition of MLC

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phosphorylation by ML-7 (a specific inhibitor of MLC kinase), suggesting PKC increases permeability through affecting actin-myosin interaction [176]. The importance of PKC activation in increasing EC permeability is that elevated glucose is known to augment PKC activation in a number of cell types, including ECs [181, 182]. Cells exposed to high glucose have an increased flux of glucose through the glycolytic pathway, causing de novo synthesis of DAG [181, 183]. DAG is the primary activator of PKC and its enhanced activation in response to elevated glucose is thought to be a contributor to many of the vascular complications associated with diabetes, including edema formation [181, 182, 184]. It is likely that enhanced PKC activity predisposes the cerebral endothelium to increased permeability, providing a mechanism by which hyperglycemia worsens postischemic edema formation. Hyperglycemia-induced PKC activation has been recognized for some time and has provided a therapeutic target for treatment of diabetic peripheral vascular disease. A similar therapeutic target likely exists for the cerebral circulation as well.

25.9 Hemodynamic Changes During Ischemia and Reperfusion and Its Role in Cerebral Edema

Ischemia is associated with significant alterations in hemodynamic forces on the cerebral endothelium that have been shown to influence vascular permeability. Flow cessation causes a decrease in both pressure and circumferential wall tension in the cerebral circulation, as well as alterations in shear stress, all of which have been shown to influence barrier properties. Reduced flow in anesthetized female rats causes both decreased pressure and transport of insulin and potassium across the BBB, suggesting that ionic homeostasis may be disturbed under conditions of reduced flow [185]. Shear stress alterations due to flow cessation have been studied in a dynamic model of BBB in which cerebral microvascular endothelial cells were cocultured with astrocytes in a tube in which flow and shear stress were controlled [10]. One hour of normoxic, normoglycemic flow cessation caused a drop in nitric oxide production that was restored upon reperfusion. These changes were not accompanied by measurable changes in TEER, suggesting that paracellular permeability was not changed during alterations in shear stress. Transcellular permeability was not measured in this study. The reperfusion period following the opening of an occluded artery is associated with autoregulatory failure and diminished myogenic tone in the cerebral arteries and arterioles, all factors contributing to reperfusion-induced hyperemia [186–188]. While hyperemia increases CBF and delivers substrates to an ischemic region, the loss of tone during this postischemic period has been shown to cause BBB disruption and edema [118, 186, 189]. The effect of postischemic hyperemia on BBB disruption and edema formation was investigated by Kuroiwa et al., who demonstrated that ischemic edema was severely exacerbated by hyperemia during reperfusion and that suppression of postischemic hyperemia

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26 Diabetes and the Consequences for the Blood-Brain Barrier Arshag D. Mooradian

26.1 Introduction

The prevalence of diabetes is rapidly increasing worldwide. This change in the epidemiology of diabetes is fuelling the surge in studies related to the pathogenesis and complications of this disease. Diabetes is a systemic metabolic disorder that affects various tissues of the body, albeit to a variable degree. At the present time, there are several potential biochemical changes that contribute to the emergence of diabetes complications. These include increased oxidative load, increased glycation of important proteins, activation of protein kinase C pathway and possibly increased accumulation of intracellular sorbitol and depletion of myoinositol [1, 2]. These biochemical changes cause, at least partly, the various long-term complications of diabetes, notably retinopathy, nephropathy, neuropathy and macrovascular disease. The central nervous system (CNS) complications of diabetes have not been widely appreciated, primarily because the most overt complication, namely stroke, is viewed as a vascular complication while the other more subtle changes in the CNS are usually not clinically relevant. Nevertheless, in the past decade there has been increasing evidence to indicate that diabetes may have significant effects on the CNS that extend beyond clinically appreciable cerebrovascular accidents [3–6]. There are several potential mechanisms that contribute to the diabetes-related changes in the CNS [5]. These include alterations in cerebral microvessels with poor auto-regulation, blood distribution, and altered blood-brain barrier (BBB) function, neurochemical changes, alterations in neurotransmitter receptor activity and other contributing factors, such as repeated hypoglycemic reactions, peripheral neuropathy and nephropathy with uremia. In this chapter, the changes in cerebral microvasculature will be reviewed and the potential clinical manifestations of diabetes-related changes in the CNS will be discussed from the perspective of the BBB function.


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26 Diabetes and the Consequences for the Blood-Brain Barrier

26.2 Histological Changes in the Cerebral Microvessels

In addition to the well characterized ischemic changes that occur following a cerebrovascular accident (see Chapter 25), the microvasculature within the CNS, like the microvasculature in a variety of tissues in a diabetic animal, undergoes significant histological changes [7–10]. Although the overall number of cerebral capillaries may not be altered [7], the density of capillaries within the cortical areas may be reduced and the capillary basement membrane thickening is increased in streptozotocin (STZ)-induced diabetic animals [8–10]. In addition, calcium depositions have been documented in microvessel walls of these animals [9]. Studies using intravital microscopy have also observed the formation of arterio-venous shunting in cerebral cortical tissue after 5 months of uncontrolled experimental diabetes [9]. These changes, although significant, are not of the same magnitude as those observed in retinal tissue where the microvasculature is also endowed with tight junctions between endothelial cells. Thus, local tissue factors appear to be an important determinant of the alterations in the microvessels that occur in diabetes. It is noteworthy that some of the histological changes in cerebral microvessels in diabetes, namely the paucity of cortical capillaries and establishment of arterio-venous shunts are similar to the changes described in aging rat models [11]. These similarities in histological changes in cerebral microvasculature support the notion that diabetes may often simulate premature aging [12]. The structural changes of cerebral microvessels predispose the animal to cerebral hypoxic injury during periods of reduced blood flow or poor ventilation and may contribute to changes in the BBB function.

26.3 Functional Changes in the Blood-Brain Barrier

The principal known functions of the BBB are to assure the adequate delivery of nutrients and substrates to the CNS, eliminating metabolic end-products through efflux and protecting the CNS from environmental toxins circulating in the blood. The latter is accomplished by the dynamic barrier that is formed by the tight junctions between endothelial cells. The molecular anatomy of these tight junctions may change in diabetes. In a recent study, Western and Northern blot analyses were carried out to measure the steady-state level of occludin and zonula occludens-1 (ZO-1) proteins and mRNA levels in cerebral tissue of STZ-induced diabetic rats and the results were compared to insulin-treated diabetic rats and vehicle-injected control rats [13]. The cerebral occludin content in diabetic rats was significantly reduced compared to insulin-treated diabetic rats or nondiabetic control rats. The ZO-1 content of cerebral tissue from diabetic rats was not significantly altered compared to controls [13]. The cerebral occlu-


din or ZO-1 mRNA content relative to G3PDH mRNA was not altered in diabetes. Thus, select structural proteins in the tight junctions may be altered in diabetes. It is noteworthy that occludin content of rat retina is reduced in experimental diabetes [14]. In addition, high glucose or low insulin concentrations reduce the ZO-1 protein content of bovine retinal capillary endothelial cells in culture [15]. Considering the similarities in the tight junctions of the BBB and blood-retinal barrier, it is not surprising that the changes in occludin content of the two vascular barriers in diabetic rats may be similar. However, the physiological consequences of these changes are not readily demonstrable. Studies on the barrier function have not always been consistent. Using immuno-histochemical techniques, Stauber et al. [16] found that albumin, but not immunoglobulin G or complement C, selectively enters the cerebral cortex after 2 weeks of experimental diabetes. In another study, extravasations of Evan’s blue albumin into the cerebral tissue of diabetic rats after adrenalin induced acute hypertension was significantly increased compared to controls [17]. Furthermore, studies using intravital microscopy and fluorescein-labeled albumin found increased BBB permeability in diabetes, although they did not find any increase in the permeability of the BBB with acute hypertension [18]. These results should be interpreted with caution since extravasation of ligands through the BBB is often hard to quantitate with intravital microscopy. The increased permeability of the cerebral microvessels to albumin is suspected to be the result of enhanced microvascular uptake of glycosylated albumin [19] and is considered to be independent of alteration in the BBB. In this regard, it is noteworthy that the BBB permeability to albumin in long-term diabetic rats could be normalized following insulin treatment [20]. The BBB permeability to a variety of markers has been studied. The volume of distribution of inulin in select regions of the cerebrum, such as mediobasal hypothalamus, mediodorsal hypothalamus and periaqueductal gray [21], is increased in rats after 4 weeks of uncontrolled hyperglycemia. However, the permeability of the cerebral microvessels to other markers such as horseradish peroxidase, sucrose, or cytochrome c is not altered in diabetic rats [21, 22]. Overall, it appears that the barrier function of the BBB in diabetic rats is well maintained, but it may be impaired, especially in certain regions of the cerebrum. Future studies should employ a host of markers with a wide spectrum of size and polarity to more comprehensively study the effect of diabetes on the BBB permeability profile.

26.3.1 Diabetes-Related Changes in the BBB Transport Function

In addition to being a protective barrier, the cerebral microcirculation plays a critical role in regulating the availability of nutrients and substrates for the production of neurotransmitters. To maintain an optimal physiologic milieu, the cerebral microcirculation regulates the efflux of byproducts of the cerebral meta-

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bolism. These transport functions are highly adaptable in response to alterations in blood glucose levels. Thus, sustained hypoglycemia or hyperglycemia is associated with significant changes in various transport parameters. One of the key alterations is the up-regulation or down-regulation of glucose transport as a consequence of chronic hypoglycemia and hyperglycemia, respectively. Some studies in human subjects have found that sustained hypoglycemia for several days induces an increase in BBB transport of glucose [23–25]. However, studies by Segel et al. [26] could not confirm these findings. In this study, there was no increase in blood-to-brain glucose transport after 24 h of experimental sustained hypoglycemia in eight healthy subjects. These investigators did not exclude the possibility of a change in regional blood-to-brain glucose transport and could not address the possibility that alterations in transport might occur beyond the BBB [26]. The effect of hypoglycemia on BBB transport function and cerebral metabolism is more comprehensively studied in animal models. In chronically hypoglycemic rats bearing insulinomas, glucose transport is up-regulated while the transport of large neutral amino acids and choline is unaffected and the transport of lactate and pyruvate is diminished [27]. Furthermore, in this rat model of hypoglycemia, cerebral tissue content of glucose-6-phosphate dehydrogenase (G6PD), creatine phosphate and ATP are not significantly decreased. This suggests that the enhanced glucose extraction at the BBB during hypoglycemia preserves brain energy consumption [27]. However, despite such adaptations to hypoglycemia [27–30], the mitochondrial respiration within the cerebral tissue remains abnormal [31]. The effect of sustained hyperglycemia on the BBB transport of nutrients has been studied in various animal models of diabetes and in human subjects with diabetes. Most, but not all studies have found that chronic hyperglycemia is associated with down-regulation of the BBB transport of glucose [32–37]. Insulin treatment with amelioration of hyperglycemia normalizes BBB glucose transport [33–35]. However, some studies have questioned this finding [36, 37]. Differences in the experimental models used and the degree or duration of hyperglycemia as well as differences in methodology employed, may explain the discrepancy in the reported results. In addition, cerebral microvessels isolated from diabetic animals have altered glucose metabolism [38]. The latter observation could have been because of reduced cellular uptake of glucose, presumably as a result of reduced glucose transporter function or could be secondary to alterations in metabolic pathways of glucose metabolism beyond the glucose transporter. There are few clinical studies on BBB glucose transport during hyperglycemia. Fanelli and his colleagues [39] found that neither blood-to-brain glucose transport nor cerebral glucose metabolism is measurably reduced in people with poorly controlled type 1 diabetes. Brooks et al. [40] observed 18% reduction in blood-to-brain glucose transport in poorly controlled diabetic patients, but this difference was not statistically significant. In a small study of six human subjects, Hasselbalch et al. [41] found that 2 h of acute hyperglycemia did not cause


significant changes in the maximal transport velocity or affinity in the glucose transporter of the BBB. In addition, a short duration of hyperglycemia did not change the global cerebral blood flow (CBF) or cerebral glucose metabolism rate (CMRglc), except in the white matter where a significant increase in CMRglc could be demonstrated [41]. Inconsistencies in the published literature on diabetes-related changes in BBB glucose transport are further compounded by the inconsistencies in studies evaluating the molecular basis of changes in BBB glucose transport. Choi et al. [42] found increased GLUT-1 mRNA levels in the brain capillaries of rats with STZ-induced diabetes but decreased transport and GLUT-1 protein (the major BBB glucose transporter). To determine the molecular mechanisms of diabetes-related changes in the expression of GLUT-1 in cerebral tissue, we studied STZ-induced diabetic rats and vehicle-injected controls after 4 weeks of diabetes [43]. The GLUT-1 mass in cerebral microvessels was reduced in diabetic rats by approximately 38%. The GLUT-1 concentration in the insulintreated diabetic group was not significantly different from controls. The GLUT-1 mRNA content of cerebral tissue in diabetic rats was significantly reduced compared to control rats or insulin-treated diabetic rats. The in vitro translation of GLUT-1 mRNA of diabetic rats was also significantly lower than that in nondiabetic control rats or insulin-treated diabetic rats [43]. These changes occurred in association with a reduction in poly(A) tail length of GLUT-1 mRNA, which decreased from a control value of 200–350 nt to only 50–100 nt in diabetic rats [43]. Shortening of the poly(A) tail of mRNAs is a novel mechanism of diabetesrelated changes in the expression of specific genes that are regulated at a translational level. In addition to the changes in BBB glucose transport, diabetes is associated with a host of changes in the transport of other nutrients and metabolites. Since the glucose transporter mediates the BBB transport of various hexoses, it is not surprising that diabetes is associated with a reduction in brain uptake of hexoses other than glucose [33]. In addition, the hexose transporter is also the key cellular transporter of dehydroascorbate [44]. In previously published studies, we demonstrated that dehydroascorbate, but not ascorbate, competes with glucose for entry into the brain [45]. Whether sustained hyperglycemia in diabetes deprives the brain of vitamin C and whether vitamin C deficiency in brain tissues leads to cerebral dysfunction is not known [45]. Nevertheless, several studies in animal models of diabetes or in human subjects have suggested that tissue deficiency in ascorbic acid may occur [46, 47], and may partially account for increased oxidative load in diabetes [48, 49]. Another example of adaptive changes in BBB related to diabetes is the increased BBB transport of ketones, such as hydroxybutyrate. This increase in ketone body transport is normalized after insulin treatment [33]. A similar up-regulation of BBB transport of ketones is reported in other models of ketosis. The exact mechanism of this change is not known. It is tempting to suggest that it may be related to alterations in the expression of ketone body transporter within the BBB. Indeed, ketosis and independently acidosis has been shown to alter

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the expression of several genes through a pH response element in the regulatory regions of genes [50]. Unlike the changes in BBB glucose and ketone transport, diabetes does not appear to alter the transport of amino acids across the BBB [33]. However, the permeability of phenylalanine was decreased by an average of 40% throughout the entire brain after 4 weeks of STZ diabetes in rats [51] and influx was depressed by 35%. The permeability of lysine was increased by an average of 44%, but the influx was decreased by 27%. Several plasma neutral amino acids (branched chain) were increased, whereas all basic amino acids were decreased. Brain tryptophan, phenylalanine, tyrosine, methionine and lysine contents were markedly decreased. The transport changes were attributed to alterations in the concentrations of the plasma amino acids that compete for the neutral and basic amino acid carriers at the BBB [51]. The BBB transport of choline, the substrate for neurotransmitter acetylcholine, is also reduced in STZ-induced diabetic rats [52]. Insulin treatment for 5 days did not rectify the abnormal choline transport. This change may contribute to the cognitive and memory changes related to diabetes [5, 6]. It is of note that the BBB choline transport is also reduced in aging animals and humans where memory deficits are common [53]. The BBB permeability to minerals such as sodium and potassium, but not chloride or calcium, is also decreased in diabetic rats [54, 55]. These changes may be related to alterations in the activity of key ATPase-dependent pumps. However, in our studies of cerebral microvascular ATPases, we found that the sodium/potassium or magnesium ATPase activity of the endothelial cells of the BBB was not altered in diabetic rats [56]. This finding was unexpected since glucotoxicity, via activation of the polyol pathway, is often associated with reduced activity of Na+/K+-ATPase [57]. However, the role of the polyol pathway in diabetes-related changes in Na+/K+-ATPase activity is not clear, since treatment of diabetic rats with an aldose reductase inhibitor does not normalize BBB permeability to sodium, although dietary supplementation with myoinositol does [55]. The effect of diabetes on the BBB transport of various lipophilic hormones or drugs is not well studied. The cerebral transport of thyroid hormone may be reduced in diabetes [58]. However, the brain uptake of benzodiazepines with different lipophilic and protein-binding characteristics is not altered in diabetes [59]. Because of conceptual importance and potential clinical implications, more studies are needed to determine whether diabetes alters the brain uptake of various centrally acting drugs. Overall, it appears that the BBB transport of certain substrates is altered by diabetes. Most studies are done in animal models of diabetes with 2–4 weeks of uncontrolled diabetes, giving results like these: 1. Barrier function · increased permeability to albumin, inulin; · unaltered permeability to immunoglobulin G, complement 3, horseradish peroxidase, ethylenediaminetetraacetic acid (EDTA), sucrose.

26.4 Potential Mechanisms of Changes in the BBB

2. Transport function · increased transport of monocarboxylic acids, e.g. beta hydroxybutyrate; · decreased transport of hexoses (e.g. glucose), dehydroascorbate, choline, sodium, potassium, protons (H+); · unaltered transport of neutral amino acids (e.g. tryptophan, tyrosine), basic amino acids (e.g. lysine), lactate, calcium, chloride. In addition, repeated hypoglycemic episodes and hyperinsulinemia in obese type 2 diabetic individuals, or insulin deficiency, may contribute to the diabetesrelated changes in BBB transport. 26.4 Potential Mechanisms of Changes in the BBB

The barrier function of the cerebral microvasculature is determined by both structural and functional characteristics, such as tight junctions between endothelial cells, the ionic charges on the surface of endothelial cells and a host of enzymes and transporters that limit the access of circulating peptides or potentially toxic substrates into the CNS. Thus, diabetes-related changes in the BBB can be the result of alterations in the hemodynamics of cerebral circulation, changes in the physico-chemical properties of key constituents of the cerebral microvessels or changes in neuronal control of the BBB. These changes are usually the result of chronic uncontrolled hyperglycemia, although co-morbidities such as hypertension [60, 61], transient cerebral ischemic events [62, 63] and hyperosmolality [64] would further aggravate the changes in the BBB. These potential mechanisms will be discussed briefly. 26.4.1 Hemodynamic Changes

Studies using intravital microscopy have found significant changes in the cerebral microcirculation of diabetic animals [65, 66]. As early as 1 month, the pial vessels of diabetic rats were dilated and tortuous and the vasoconstrictive response of arterioles and venules to local increases of PO2 in the artificial cerebrospinal fluid (CSF) bathing these vessels was reduced [9]. In addition, the linear velocity of blood flow was reduced in diabetic rats and increased arterio-venous shunting was also observed. Electron microscopic studies found that, after 5 months of diabetes, focal changes in the thickness and density of the vascular basement membrane were noted [9]. In addition, astrocytic endfeet were swollen and contained mitochondria having a longitudinal rearrangement of their cristae. Basement membranes contained nodules of electron-lucent material that impinged on degenerating smooth muscle cells, pericytes and astrocytes. The tight interendothelial junctions appeared to be intact even though dramatic changes had taken place perivascularly. Although the vasoconstrictive response to in-

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creased PO2 is reduced in diabetes, the constriction of pial arterioles in response to norepinephrine, serotonin, prostaglandin F2 alpha and thromboxane analog is not altered [65–68]. Likewise, the dilation of cerebral arterioles in response to papaverine or nitroglycerine was not significantly altered in those studies. These changes in vasomotor responsiveness and, more importantly, the establishment of arteriovenous shunts would adversely affect the delivery of nutrients to cerebral tissue.

26.4.2 Biophysical and Biochemical Changes

Microviscosity of endothelial cell membranes is an important determinant of transport across the BBB. This parameter is modulated by the lipid and protein composition of the membranes. Experiments in endothelial cells isolated from diabetic rats have not shown significant alteration in membrane viscosity measured with two probes: one that localizes to the surface of the membrane and another that localizes in the core of the membrane [69]. This observation is in agreement with the lack of significant changes in membrane content of the major determinants of microviscosity, namely cholesterol, phospholipids and fatty acid composition [69]. However, the concentration of lipid peroxidation byproducts, as measured by conjugated dienes or malondialdehyde modifications of proteins, are increased in the cerebral microvessels of diabetic rats [69]. The effect of diabetes on membrane microviscosity is variable. Although significant changes in red cell membranes have been reported [70], there are no significant changes in the microviscosity or lipid composition of synaptosomal membranes after 6 weeks of STZ-induced diabetes [71]. The increased lipid peroxidation byproducts within the cerebral microvessels of diabetic animals may well be related to an altered antioxidant defense system. Thus, the antioxidative potential of cerebral microvessels, measured in the presence of a peroxy radical generator, is reduced in diabetic rats [72]. In addition, increased oxidative stress is well documented in diabetes [48]. This increase in oxidative load could be the direct result of hyperglycemia and glyco-oxidation [48, 49, 73], or could be secondary to a decrease in antioxidant activity [46, 47]. In addition to the modest changes in lipid composition observed in the cerebral microvessels of diabetic rats, there are some changes in the protein composition. Analysis of the protein content of cerebral microvessels with two-dimensional gel electrophoresis revealed that only one out of 25 proteins quantitated was significantly altered in diabetic rats after 5 weeks of uncontrolled hyperglycemia [74]. In addition, post-translational modifications of proteins such as glycation and malondialdehyde modification have been documented [74]. Diabetes also alters the content or activity of select proteins in the cerebral microvessels. A potentially important structural protein that is found within the cerebral microvessels is the endothelial barrier antigen (EBA). In an in situ quantitative study, the EBA content of the BBB, relative to glucose transporter 1

26.4 Potential Mechanisms of Changes in the BBB

(GLUT-1) was not altered in diabetes [75]. However, since GLUT-1 may be reduced in diabetic rats, it is likely that EBA is also reduced in diabetes. The activity of a host of enzymes within the cerebral microvessels may also be altered in diabetes. The adenylate cyclase activity of cerebral microvessels is reduced in diabetic rats without a change in beta-adrenergic receptor number or receptor-cyclase coupling [76]. However, in another study, the beta-adrenergic receptor number was found to be reduced [77]. These changes have important physiological implications, since blood flow through cerebral microvessels is regulated by neurotransmitter activity [78–80] and the dilatory effect of betaadrenergic receptor activation on large and small cerebral vessels is impaired in diabetics [81, 82]. The sodium/potassium or magnesium ATPase activity of the endothelial cells of the BBB was not altered in diabetic rats [56]. This finding was unexpected, since glucotoxicity via activation of polyol pathway is often associated with reduced activity of Na+/K+-ATPase [57]. The vacuolar type H+-ATPase, and not the gastric H+/K+-ATPase, is present in cerebral tissue, including cerebral microvessels and choroid plexus [83]. The functional role of the vacuolar H+-ATPase in the BBB remains to be determined. The quantity of vacuolar H+-ATPase in the BBB is modulated by blood H+ or HCO–3 content. These changes may be relevant to the physiology of the acid-base balance in the CNS. Diabetic rats have reduced ATPase inhibition by N-ethylmaleimide (NEM), a vacuolar proton (H+)-ATPase inhibitor [84]. The changes in cerebral microvessels were distinct from those found in synaptosomal membranes, indicating the cell specificity of the findings. Overall, it appears that diabetes may well be associated with structural changes in specific proteins in cerebral microvessels. Both qualitative and quantitative changes in protein composition may play a significant role in diabetesrelated alteration in the BBB.

26.4.3 Changes in Neurotransmitter Activity in Cerebral Capillaries

In addition to the potential changes in beta-adrenergic neurotransmission through the adenylate cyclase system mentioned above, other changes in the neurotransmitter activity of cerebral capillaries may occur. For example, glutamate has been implicated in the BBB permeability to low molecular weight ligands. This effect may be mediated by nitric oxide (NO)-dependent mechanisms [85]. NO and endothelin-1 (En-1) are found to alter the BBB function, but their role in diabetes-related changes of the BBB is still not known [86, 87]. NO production increases the gamma-amino butyric acid permeability of the BBB [88], while endothelin receptor-mediated activation of protein kinase C (PKC) increases the permeability of the BBB for low molecular weight ligands and may contribute to the formation of cerebral edema [89]. In cultured rat brain capillary endothelial cells, endothelin-1 and -3 can affect the activities of ion and water transport at the BBB

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through different signal transduction mechanisms [90]. Endothelins stimulate sodium uptake into rat brain capillary endothelial cells through endothelin A-like receptors [91]. Some of these effects of endothelin are mediated through phosphorylation of key proteins in endothelial cells [92]. Multiple signaling pathways are involved in the regulation of tight junction permeability [93, 94]. These include the cyclic nucleotides that decrease paracellular permeability, protein kinase C, G proteins, MAP kinases and other protein kinases. The effect of diabetes on these signaling pathways within the cerebral microcirculation is not well characterized.

26.5 Potential Clinical Consequences of Changes in the BBB

A host of diabetes-related changes in the CNS has been recognized. The pathogenesis of CNS diseases is often related to cerebrovascular accidents, repeated hypoglycemic attacks, ketosis, hyperglycemia and hyperosmolarity. In addition, peripheral or autonomic neuropathy, impaired vision, renal failure and medications further aggravate CNS-related diseases in diabetes [3–5]. Although there are multiple potential causes underlying these changes, the BBB function is likely to be an important contributor to the changes in the CNS. Clinical evidence for loss of BBB integrity in diabetic subjects is scarce. This is the result of the limitations in the current technology available to study the BBB function in human subjects. In this context, the availability of reliable serum biomarkers of the barrier function would be helpful. One such marker of BBB openings is the serum level of S100B protein and/or the presence of anti-S100B auto-antibodies [95–97]. The S100B is an astroglial protein that belongs to the calcium receptor superfamily. Serum levels of S100B protein are increased when BBB is disrupted with mannitol [96]. When the BBB disruption is severe, neuronal injury is reflected in the increased levels of serum neuron-specific enolase (NSE) [96]. The measurement of serum concentrations of NSE and S100B may have a prognostic role in evaluating clinical outcome following severe hypoglycemia [98]. In one case of diabetic ketoacidosis, serum S100B concentration rose coinciding with the onset of cerebral edema [99]. To determine whether clinical diabetes is associated with disruption of the BBB and/or brain injury, serum levels of S100B and NSE as well as auto-antibodies against them were measured in subjects with type 1 and type 2 diabetes [100]. Serum S100B concentrations in type 2 diabetic subjects, but not in type 1 diabetic subjects, were significantly lower than those found in healthy controls. There were no significant differences in serum NSE levels of either type 1 or type 2 diabetics compared to healthy controls. However, there was a significant increase in antibodies to NSE in both type 1 and type 2 diabetic subjects compared to controls, whereas diabetics and controls had equally very low levels of anti S100B auto-antibodies. These studies suggest that diabetes in humans may be associated with alterations in the BBB integrity that allow the emergence of antibodies against neuronal antigens [100].

26.5 Potential Clinical Consequences of Changes in the BBB

Cerebrovascular accidents are widely recognized as the most serious complication of diabetes. However, diabetes is also associated with less dramatic disease of the CNS, including the slow deterioration of cognitive function, increased incidence of depression and reduced pain tolerance. Several clinical studies have found that type 1 diabetic patients have modest impairment in learning, memory and problem solving, as well as impairments in mental and motor speed [101, 102]. The cognitive changes of type 2 diabetic patients are more consistently demonstrable. Moderate cognitive impairment is reported particularly in tasks involving verbal memory and complex information processing, while basic attention process, motor reaction time and short-term memory are relatively unaffected [101, 102]. Several studies have reported that diabetic patients are significantly inferior to the nondiabetic control group in the serial learning task and Benton’s visual retention test [103–105]. The digit span test usually shows no significant difference between the groups. Electroencephalogram (EEG) frequency band analysis has shown a slowing over the central cortex and reduction of alpha activity over the parietal area in diabetic patients [105]. The checkerboard-elicited P100 wave did not reveal a significant increase in latency nor were the P300 wave latencies significantly different in diabetic patients. However, a trend toward longer latencies in diabetics was evident at Fz and Cz recording sites [105]. These observations suggest that diabetic patients have an impairment in the retrieval of recently learned material with preservation of auditory attention and immediate recall. It is also possible that the diabetes-related changes in the BBB contribute to the increased incidence of Alzheimer’s disease in diabetes [106]. The changes observed in diabetes are often similar to those found in aging [107]; and disruptions in the BBB have been suspected to be a contributing cause of Alzheimer’s disease [108]. Indeed, BBB changes in the GLUT-1 content of cerebral tissue from patients with Alzheimer’s disease are similar to those found in diabetes [109]. Similar changes in cognition are found in animal models of diabetes [110, 111]. In one study, mice with STZ-induced diabetes mellitus had normal acquisition for relatively simple tasks but showed problems in learning more complex tasks such as shuttle box avoidance. However, other studies have found increased learning in diabetes. In the latter studies, enhanced learning shown in simple passive avoidance tasks appears to be due to increased foot-shock sensitivity [112]. Diabetic mice also show a marked memory retention deficit after learning an active avoidance T-maze task. This retention deficit was reversed by a single injection of insulin, suggesting that it may be related to hyperglycemia per se. Furthermore, diabetic mice were found to have a shift to the left in the inverted U-shaped dose-response curve for memory retention produced by an acetylcholine agonist [113]. Responses to several other pharmacological memory enhancers are also altered in diabetic mice. These studies suggest that this mouse model of diabetes mellitus demonstrates a deficit in memory retention and retrieval similar to that seen in humans with diabetes mellitus. In addition to cognitive changes in diabetes, affective disorders and notably the incidence of depression are increased in diabetes [114]. Major depression

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has a higher recurrence rate and depressive episodes may last longer in individuals with type 1 or type 2 diabetes [115–119]. It is not entirely clear whether this is the result of neurobiochemical changes associated with diabetes, or the result of psychological factors related to chronic disease state or its treatment. It is noteworthy that depressive disorders may increase the risk for diabetes, possibly because of increased insulin resistance presumably secondary to increased plasma corticosteroid levels [120]. The onset of major depression typically precedes the diagnosis of type 2 diabetes by many years, while in type 1 diabetes it seems to follow the diagnosis [115–119]. A significant relationship between poor glycemic control and major depression has been reported, although this relationship was not supported by other studies [120–123]. Another clinical manifestation of diabetes-related changes in the CNS is alteration in pain perception. Pain intolerance is a common feature of diabetes, independent of peripheral neuropathy [124, 125]. Animal studies have suggested an altered response to opiate agonists and antagonists as well as an altered pain threshold in diabetic animals [126]. STZ-induced diabetic rats have a higher spontaneous pain threshold but reduced sensitivity to morphine analgesia [126]. The exaggerated painful response to stimuli, as well as spontaneous pain in diabetic rats was attributed to hyper-responsiveness and increased conduction veloci-ty of a subset of C-nerve fibers [126]. In clinical studies, infusion of 50 g glucose in normal subjects resulted in a significant decrease in both the threshold level of pain and the maximal level of pain tolerated, as measured by responses to electrical pain induced by a Grass stimulator [127]. In addition, patients with diabetes mellitus were hyperalgesic when compared with normal subjects. These findings have potential clinical implications in the pathophysiology and management of painful diabetic neuropathy and the use of narcotic agents in diabetes mellitus. The sensory threshold of pain is elevated in diabetic subjects. Major risk factors for the elevation of sensory threshold for pain were age, duration of diabetes and tall stature [127]. Finally, diabetic subjects have a prolongation of anginal perceptual threshold (the time from the onset of 0.1 mV ST segment depression to the onset of chest pain during treadmill exercise). This prolongation correlates positively with the somatic pain threshold [125]. The changes in anginal threshold may delay the diagnosis of myocardial infarction in these patients. Another potential consequence of the changes in BBB is decreased responsiveness to certain hormones, notably leptin [128]. It appears that obesity-related resistance to leptin is partially the result of reduced transport of leptin across the BBB in obese individuals. Finally, it is possible that the aggravation of hypoglycemia unawareness in individuals with long-standing diabetes as a result of very strict blood glucose control [129] may partly be related to up-regulation of the glucose transporters in response to repeated hypoglycemic attacks [27]. The up-regulation of glucose transporters in these patients may theoretically retard the CNS response to hypoglycemia. Alternatively, the down-regulation of glucose transporters in chronic hyperglycemia may account for the emergence of

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26.6 Conclusions

These observations indicate that uncontrolled hyperglycemia significantly alters the biochemical composition and physiology of cerebral microvessels. These alterations may have subtle clinical implications. It is possible that the down-regulation of glucose transport at the BBB may protect the CNS against the ravages of hyperglycemia. There are multiple mechanisms involved in the changes observed at the BBB. The relative importance of these mechanisms to the overall alterations in BBB function is not known. Future studies should address the unique responses of the BBB to hyperglycemia and altered metabolic milieu commonly seen in diabetes. In addition, pharmacologic interventions that can protect the BBB function independent of the degree of hyperglycemic control would help ameliorate some of the complications of diabetes.

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Cortical function in elderly non-insulin dependent diabetic patients: behavioral and electrophysiological studies, Arch. Int. Med. 148, 2369–2372. D. Galasko 2003, Insulin and Alzheimer’s disease: an amyloid connection, Neurology 60, 1886–1887. G. N. Shah, A. D. Mooradian 1997, Age-related changes in the blood-brain barrier, Exp. Gerontol. 32, 501–519. M. O. Chaney, J. Baudry, C. Esh, J. Childress, D. C. Luehrs, T. A. Kokjohn, A. E. Roher 2003, Diabetes, aging, and Alzheimer’s disease: a tale, models, and hypotheses, Neurol. Res. 25, 581–589. A. D. Mooradian, H. C. Chung, G. N. Shah 1997, GLUT-1 expression in the cerebra of patients with Alzheimer’s disease, Neurobiol. Aging 18, 469–474. J. F. Flood, A. D. Mooradian, J. E. Morley 1990, Characteristics of learning and memory in streptozotocin-induced diabetic mice, Diabetes 39, 1391–1398. G. J. Biessels, A. Kamal, I. J. Urban, B. M. Spruijt, D. W. Erkelens, W. H. Gispen 1998, Water maze learning and hippocampal synaptic plasticity in streptozocin-diabetic rats: effects of insulin treatment, Brain Res. 800, 125– 135. J. A. Gavard, P. J. Lustman, R. E. Clouse 1993, Prevalence of depression in adults with diabetes, Diabetes Care 16, 1167–1178. P. J. Lustman, L. S. Griffith, R. E. Clouse 1988, Depression in adults with diabetes: results of a 5 year follow-up study, Diabetes Care 11, 605–612. P. J. Lustman, L. S. Griffith, R. E. Clouse, K. E. Freedland, S. A. Eisen, E. H. Rubin, R. M. Carney, J. B. McGill 1997, Effects of nortriptyline on depression and glycemic control in diabetes: results of a double-blind, placebocontrolled trial, Psychosom. Med. 59, 241–250. P. J. Lustman, L. S. Griffith, K. E. Freedland, R. E. Clouse 1997, The course of major depression in diabetes, Gen. Hosp. Psychiatry 19, 138–143. M. Kovacs, D. S. Obrosky, D. Goldston, A. Drash 1997, Major depressive disorder in youths with IDDM: a controlled prospective study of course and outcome, Diabetes Care 20, 45–51. S. Kalmijn, E. J. Feskens, L. J. Launer, T. Stijnen, D. Kromhout 1995, Glucose tolerance, hyperinsulinemia and cognitive function in a general population of elderly men, Diabetologia 38, 1096–1102. P. J. Lustman, L. S. Griffith, R. E. Clouse, P. E. Cryer 1986, Psychiatric illness in diabetes mellitus: relation to symptoms and glucose control, J. Nerv. Ment. Dis. 174, 736–746. M. Peyrot, R. R. Rubin 1999, Persistence of depressive symptoms in diabetic adults, Diabetes Care 22, 448–452. S. J. Niemczyk, M. A. Speers, L. B. Travis, H. E. Gary 1990, Psychosocial correlates of hemoglobin A 1c in young adults with type I diabetes, J. Psychosom. Res. 34, 617–627. A. M. Jacobson, A. G. Adler, J. I. Wolfsdorf, B. Anderson, L. Derby 1990, Psychological characteristics of adults with IDDM: comparison of patients in poor and good glycemic control, Diabetes Care 13, 375–381.

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27 Human Parasitic Disease in the Context of the Blood-Brain Barrier – Effects, Interactions, and Transgressions Mahalia S. Desruisseaux, Louis M. Weiss, Herbert B. Tanowitz, Adam Mott, and Danny A. Milner

27.1 Introduction (by D. Milner)

Human parasitic diseases currently comprise the largest group of infections in humans worldwide including the nematodes (“roundworms”) at *3.7 ´ 1012, trematodes (“flatworms” or “flukes”) at > 200 ´ 106, cestodes (“tapeworms”) at > 50 ´ 106, the protozoa at > 500 ´ 106, and amoeba at > 50 ´ 106. Although the population at risk for certain diseases such as malaria has decreased over the past 40 years, the number of individuals infected has increased, as has the mortality rate. With the exception of dracunculiasis (i.e. guinea worm), the incidence and prevalence of the vast majority parasitic diseases has steadily grown with the population, although accurate estimates of these diseases are difficult at best. The growing problem of human parasitic diseases is largely one of socioeconomic inadequacies, compounded by specific environmental and political situations. The evolution of drug resistance complicates the treatment of malaria (i.e. Plasmodium falciparum malaria, rapidly evolving resistance to a regiment of drugs), but the bulk of parasites (e.g. the intestinal nematodes, the filaria worms, the trematodes) remain susceptible to existing treatments, but require recurrent treatment and aggressive management plans if eradication is desired. When programs of prevention and elimination have been successful, the incidence and prevalence of some extremely common diseases have dropped drastically (e.g. dracunculiasis). Conversely, parasitic diseases have emerged as a result of the rising population of immunocompromised patients; and these carry higher mortality due to the lack of knowledge and drug treatments available (e.g. disseminated amebiasis). Parasitic diseases have, almost by definition, evolved to survive in harmony with their hosts. However, some degree of host morbidity is often a consequence of this coexistence and is a leading cause of massive economic loss worldwide (e.g. chronic anemia in China from hookworms, blindness in Africa from river blindness). Host mortality is a negative result for the parasite and, thus, stems from inappropriate or abnormal perturbations of the parasite’s life Blood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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27 Human Parasitic Disease in the Context of the Blood-Brain Barrier

cycle. In some cases, humans are not the intended (or usual biological) hosts of infecting parasites, but rather an inappropriate or inadequate host, and this can lead to severe consequences (e.g. African trypanosomiasis, cysticercosis). In most cases, however, the human-parasite relationship appears to be perturbed (e.g. immunocompromise, human and parasite genetic influences, other infections), resulting in fatal outcomes (e.g. toxoplasmosis, severe malaria). Central nervous system involvement by parasitic diseases, especially infections which affect, disrupt, and/or destroy the blood brain barrier are more commonly fatal than parasitic infections which do not involve the central nervous system. Overall, death of the host in parasitic diseases is a relatively rare event when compared to the pool of infected individuals. Tables 27.1 and 27.2 summarize the majority of parasites interacting with the blood-brain barrier either as part of their normal life cycles or as part of human disease. The life cycles of human parasitic diseases deserve ample attention in order to clarify why disease of the central nervous system (CNS) and breakdown of the blood-brain barrier (BBB) occur, given the prevailing concept of ultimate parasite survival. As the integrity of the CNS and the BBB are important for host survival, it seems unlikely that a disease process impairing either of these would serve an evolutionary advantage for the parasite. Figure 27.1 is a diagrammatic representation of life cycles for the majority of human parasites. Many parasitic organisms are transmitted to humans by the bites or fecal exposure near a bite from mosquitos, biting flies, and other arthropods such as Ixodes Table 27.1 Organisms affecting the blood-brain barrier. Organism type

Genus and species

Amoeba

Acanthamoeba spp Balamuthia mandrillaris Entamoeba histolytica Naegleria fowleri

Cestode

Taenia solium

Nematode

Angiostrongylus cantonensis Onchocerca volvulus Strongyloides stercoralis Toxocara canis Trichinella spp

Protozoa

Leishmania spp Plasmodium falciparum Toxoplasma gondii Trypanosoma brucei gambiense, T. b. rhodesiense Trypanosoma cruzi

Trematode

Schistosoma mansoni, S. haematobium, S. japonicum

Uncommon but worldwide

Granulomatous amoebic encephalitis (and/or disseminated disease); “contact” conjunctivitis

Eosinophilic meningitis

Granulomatous amoebic encephalitis

Amoebiasis, disseminated amoebiasis

Acanthamoeba spp (A. culbertsoni, A. polyphaga, A. castellanii, A. healyi, A. astronyxis, A. hatchetti, A. rhysodes)

Angiostrongylus cantonensis

Balamuthia mandrillaris

Entamoeba histolytica

Luminal (asymptomatic/symptomatic) is common worldwide; extraluminal (especially CNS involvement) is rare

100 cases reported worldwide

Rare (majority in Southeast Asia and Pacific Basin)

Prevalence/incidence/mortality

Disease

Organism

Table 27.2 Parasitic diseases known to affect the blood-brain barrier (all data 2005, www.cdc.gov).

Spectrum includes asymptomatic carriage, severe intraluminal intestinal disease, severe extraluminal disease with abscess of the liver and CNS

Spectrum includes granulomatous amoebic encephalitis; nasopharyngeal, cutaneous, and disseminated infections

Mostly a product of the host response to the parasite but larvae may be found in the brain; rarely fatal and usually does not require treatment unless CNS symptoms are severe

“Contact” conjunctivitis (from contaminated contact lenses) usually treatable; disseminated disease (especially in immunocompromised) almost universally fatal

Clinical outcome

27.1 Introduction 673

350 million at risk worldwide with 1.5 million cases per year of cutaneous leishmaniasis and 500 000 cases per year of visceral leishmaniasis

Leishmaniasis (Kala-azar)

Primary amoebic meningoencephalitis Onchocerciasis (river blindness)

Cerebral malaria and severe malarial anemia (severe malaria)

Leishmania spp: L. donovani complex (L. donovani, L. infantum, L. chagasi); L. mexicana complex (L. mexicana, L. amazonensis, L. venezuelensis); L. tropica; L. major; L. aethiopica; and subgenus Viannia [L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) panamensis, and L. (V.) peruviana]

Naegleria fowleri

Onchocerca volvulus

Plasmodium falciparum

2.5 billion at risk, annually 300–500 million infections, 1.5–2.0 million deaths

17.7 million infections (99% in Africa), 270,000 blind, 500,000 with visual impairment

< 30 cases in United States, but worldwide


Disease

Organism

Table 27.2 (continued)

Mortality is 20–50% for severe malaria (approximately 1.5–2.5 million deaths yearly)

Patients may first develop skin rash and subcutaneous nodules (onchocercomas) which contain adult female worms; eye lesions develop when microfilaria from gravid female begin circulating in the bloodstream and demonstrate a tropism for ocular tissues; disease is almost never fatal

Due to brain destruction, rapid progression with degenerating clinical signs; almost universally fatal

Manifestations of leishmaniasis are dependent on the species. Cutaneous leishmaniasis causes sores on the skin where sandflies have fed with localized lymphadenopathy and often spontaneous resolution. Visceral leishmaniasis results in fever, weight loss, and hepatosplenomegaly. Rarely, leishmania can cross the blood brain barrier

Clinical outcome

674


Disease Schistosomiasis (Bilharzia), neuroschistosomiasis

Strongyloidiasis, hyperinfection

Taeniasis, cysticercosis, neurocysticercosis

Organism

Schistosoma mansoni, S. haematobium, and S. japonicum

Strongyloides stercoralis

Taenia solium


Estimated to 50 million worldwide; prevalence higher in regions consuming pork; approximately 1–2% of cases develop neurocysticercosis

70 million worldwide; severe disease (hyperinfection), although very rare, has mortality up to 80%

200 million infected worldwide


Cysticercosis: dissemination of larval cysts throughout the body, usually asymptomatic (80%) unless trauma, involution, or mass effect brings them to clinical attention. Neurocysticercosis: cyst involving the central nervous system which are usually asymptomatic unless trauma, involution, or progression to mass effect brings them to clinical attention usually with seizures, hydrocephalus, and disability

Acute/chronic strongyloidiasis manifests with cutaneous, pulmonary or gastrointestinal symptoms; severe disease (immunocompromised) leads to dissemination including the CNS

Typical infections involves migration of adult worms to species specific human venous regions with production of eggs which migrate through tissues locally; rarely, eggs migrate to the brain and spinal cord resulting in neuroschistosomiasis

Clinical outcome


Disease Ocular larva migrans and visceral larva migrans

Acute localized lymphadenopathy, congenital toxoplasmosis, cerebral toxoplasmosis

Trichinellosis, trichonosis

Organism

Toxocara canis

Toxoplasma gondii

Trichinella spp (T. spiralis, T. pseudospiralis, T. nativa, T. nelsoni, T. britovi)


Worldwide; prevalence decreasing due to meat regulations; less than 15 cases yearly in United States

60 million (US data) but worldwide distribution

Worldwide; in United States, 10,000 cases occur yearly with 700 individuals developing eye damage


Infections may be asymptomatic but intestinal invasion may have diarrhea, abdominal pain, and vomiting. Muscular larval migration causes periorbital and facial edema, conjunctivitis, fever, myalgias, rashes, and eosinophilia. Fatal manifestations may include myocarditis, central nervous system involvement, and pneumonitis

Immunocompetent: rarely “flu-like” symptoms with swollen lymph nodes. Immunocompromised: acute or reactivation causes serious (often fatal) disease of the CNS. Congenital: most asymptomatic at birth with CNS problems developing later, small per cent with serious damage at birth

OLM occurs when a microscopic worm enters the eye; it may cause inflammation and formation of a scar on the retina. Symptoms of VLM, which are caused by the movement of the worms through the body, include fever, coughing, asthma, or pneumonia.

Clinical outcome

676


Disease West African [T. (b) gambiense] and East African trypanosomiasis [T. (b) rhodesiense], “African sleeping sickness” American trypanosomiasis (Chaga’s disease)

Organism

Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense

Trypanosoma cruzi


Acute (1%) can result in death with CNS involvement; chronic (20–30%) results in systemic muscular damage (e.g. cardiac, esophagus, bowel)

Universally fatal without medical treatment

40 000–100 000 new cases yearly in Africa

16–18 million infected worldwide (majority South America) with 50 000 deaths yearly

Clinical outcome



678


ticks (Fig. 27.1 A). Examples include the human malarias (Plasmodium falciparum, P. vivax, P. ovale, P. malariae) and the nematodes responsible for the human filarial infections. The overwhelming majority of these infections do not result in a fatal outcome (e.g. less than 1% for malaria), though disabling morbidity is common. A recurring theme of these infections is that no known animal reservoir exists – humans are the only host. Clinically significant disease, therefore, manifests either rarely (e.g. in P. falciparum malaria probably due to a combination of factors including human genetics, parasite burden, immune status, comorbidities) or with only great morbidity but limited mortality (e.g. lymphatic filariasis when the worm burden and inflammatory component reach a threshold). This is in stark contrast to diseases carrying large animal reservoirs (Fig. 27.1 B) such as African trypanosomiasis, in which case infections in humans are fatal if untreated, while cattle may remain asymptomatic. Other parasites with a very similar life cycle to Trypanosoma and Plasmodia, namely American trypanosomiasis and Babesia spp, have rare or little clinical consequence for the CNS. Toxoplasma gondii and Echinococcus spp are excellent examples of a life cycle where humans are incidental “end hosts” which cannot transmit the infection (Fig. 27.1 C). In toxoplasmosis, disease occurs either acutely in the infection (congenital toxoplasmosis or focal lymphadenitis) or as a result of reactivation during a period of immunosuppression (cardiac or neural toxoplasmosis). Echinococcosis results from human incidental exposure to canine or other carnivorous feces, with development of intrahost infecting cysts, which can rarely invade the central nervous system. Other less problematic diseases of a similar pattern include North American Brugia, Anisakis spp ingestion, sparganosis, Toxocara canis infection, and Dirofilaria immitis infection. The trematodes such as Schistosoma spp demonstrate yet another life cycle pattern (Fig. 27.1 D) in which humans become infected with a free-swimming stage (i.e. cercariae), harbor the worms to adulthood, and release eggs into the environment. At that point, secondary hosts (e.g. snails) are infected by the miracidia from eggs and ultimately release the sporocysts, which become the cercariae. A vulnerable point in all of these life cycles is the water source; and in each case, water purification (as was used for the nematode Dracunculus medinensis globally) and/or snail extermination (as was implemented in China under Mao-Tse Tong for the Schistosoma spp) could eradicate transmission. The amoebae and geohelminths (i.e. ascariasis, hookworms, whipworms, Strongyloides stercoralis) are examples of a continuous life cycle (Fig. 27.1 E), in which the parasitic organisms are either somewhat or completely free-living and therefore can continually reinfect humans. The vast majority of these organisms, with the exception of Entamoeba histolytica, do so with “innocent” colonization of the human host (i.e. remaining within the gastrointestinal and/or respiratory tract, without wandering). The other clinically significant amebic (discussed below) and geohelminth infections (e.g. the hyperinfection of S. stercoralis) are almost always the result of immunosuppression. Lastly, there are those parasites (e.g. cysticercosis with Taenia solium) which are usually in cyclic harmony with the host and reservoir but can enter the

27.1 Introduction

Fig. 27.1 Sets of diagrams are depicted, representing the general patterns of life cycles seen in parasites, which can interact with the blood-brain barrier. See text for details.

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680


cycle abnormally (e.g. consumption of fecal material of pigs or humans rather than consumption of infected uncooked pig meat), resulting in dissemination (Fig. 27.1 F). The spectrum of parasite interactions with the BBB is well characterized for several organisms that will be explored in more detail below. Little is known about the majority of the remaining parasite-human interactions, but a review of the anatomic pathology findings as well as the indirect evidence of involvement are helpful in understanding the resulting human diseases that are seen clinically. The anatomic pathology of the CNS as observed under light microscopy is also a useful tool for understanding the degree to which the BBB is involved by these parasitic infections and why treatment at such stages is often futile. Figure 27.2 depicts several examples of different types of pathology encountered. Plasmodium falciparum with its unique ability to sequester in human tissue can be found to “stuff” and distend cerebral vessels in patients dying of cerebral malaria. The majority of the life cycle from early trophozoites to late schizonts (Fig. 27.2 A) occurs in close proximity to human endothelium as part of every human infection (see Section 27.2 “Malaria”, below). The rate of clinically evident cerebral involvement, however, remains very rare, provoking one of the most frequently argued/long-standing controversies in the malaria community. Both African and American trypanosomiasis can been visualized by blood smear for clinical diagnosis of the infections (Fig. 27.2 B). The central nervous system pathology of the lesions are quite distinct, with African trypanosomiasis causing meningeal and perivascular mononuclear infiltration, reactive gliosis, and the classic morula cells, while American trypanosomiasis demonstrates microglial nodules and occasionally amastigotes within glial cells. Toxoplasma gondii remains quiescent in human tissues after primary infection, which can include the acute syndromes of either localized lymphadenitis in nonpregnant humans or the catastrophic infection of gestating infants seen in mothers exposed to the organism for the first time in pregnancy. When patients become immunosuppressed, particularly with falling T-cell counts, the bradyzoites (Gr. slow animals) resting within cysts reactivate to tachyzoites (Gr. fast animals), resulting in minimal inflammation, tissue necrosis, and neurologic sequelae (Fig. 27.2 C). Treatment, therefore, is directed at prevention by monitoring the serology/exposures of expectant mothers and prophylactic drug use in the immunosuppressed. The free-living amoebae generally cause massive tissue necrosis, resulting in large abscess cavities, as with Entamoeba histolytica (seen in Fig. 27.2 D with an ingested red blood cell), or diffuse granulomatous inflammation of the cerebral cortex, as with Acanthamoeba spp (seen in Fig. 27.2 E at the border of the granular and molecular layers of the cerebellum). Presentation with most amoebic infections is late, hence the high clinical failure rate in the immunosuppressed. Clinical disease and the resulting pathology of trematode infections depend on the venous bed to which the parasite couple localizes. The trematodes do not usually directly invade the CNS, but occasionally the eggs metastasize to the spinal cord and rarely to the cerebral cortex. Schistosoma haematobium male and

27.1 Introduction

female adult worms can be seen coupling within a bladder vessel, while their eggs (higher magnification inset) move towards mucosal surfaces, to return to the environment inducing a surrounding inflammation (Fig. 27.2 F). Traditionally, drugs targeting these parasites only kill the mature adult worms and thus must be taken within a precise window after known exposure to present egg-related disease. Recent data from several clinical trials demonstrates effective preventative treatment shortly after exposure for all three species with subsequent reduction in disease burden and clinical symptoms [127–129]. Cysticercosis results when humans ingest the proglottids and/or eggs of Taenia solium rather than the infected meat of a pig. In this situation, the human becomes akin to the pig, and human muscle can be encysted with metacestode larvae (Fig. 27.2 G). These viable larval cysts are almost always clinically silent unless trauma or mass effect reveals them. Conversely, when the larva dies, the immune system responds dramatically and the resultant inflammation (Fig. 27.2 H) can be a focal point of neurologic symptoms, most commonly seizures. As these signs are of dead worms, the role for parasite treatment is minimal while symptomatic treatment is paramount. The majority of filarial diseases manifest due to the adult worm burden in the host. River blindness, however, results from relatively few adult worms of Onchocerca volvulus, often embedded within a subcutaneous nodule and filled with microfilaria (Fig. 27.2 I) releasing massive amounts of progeny into the bloodstream and lymphatics. These microfilariae migrate into the eye tissues through the BBB and die. Treatment with ivermectin only suppresses the microfilarial (or larval) stages to prevent blindness while subcutaneous nodulectomy offers a definitive but disfiguring cure. Indirect evidence of CNS invasion by demonstration of the production of IgG within the cerebrospinal fluid has been seen in cysticercosis, schistosomiasis, onchocerciasis, and granulomatous amoebic encephalitis [1–9]. These findings suggest parasite antigen exposure to the responding human immune system within the CNS (and, thus, organisms having crossed the BBB). Perhaps one finds this a logical conclusion, as the pathology of these diseases (shown above) can involve large and often destructive central nervous system lesions. However, cerebrospinal fluid markers of disease prove very helpful in making definitive diagnoses, especially in locations where imaging is not available and coinfection is expected. Additional clinical markers, which are directly related to violation of the BBB by the invading parasite, include tissue-specific cytokines and neuronspecific enolase, as demonstrated by Taenia solium infection [10–13]. Although these are not specific, within the correct clinical and radiographic context, they prove extremely useful for diagnostic purposes. Other parasitic organisms may venture to the central nervous system without harm to the host. For example, in experimental mouse models, Leishmania spp were demonstrated to cross the BBB. Transgression by the same organism has been definitely reported in human patients [14, 15]. These findings are of interest because the human clinical disease of leishmaniasis does not include clinical manifestations of CNS involvement. Other disease conditions, such as the eosinophilic meningitis of An-

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682


Fig. 27.2 This set of histologic images depicts the patterns of injury seen in the central nervous system and elsewhere resulting from parasitic infections in humans. These images are hematoxylin and eosin stained sections taken under the following relative magnifications: (A) 1000 ´ in frontal lobe, (C) 1000 ´ in cerebellum, (D) 1000 ´ in cerebral cortex, (E) 400 ´ in cerebellum, (F) 20 ´ in bladder wall (inset, 400 ´ in bladder submucosa), (G) 20 ´ in skeletal muscle, (H) 20 ´ in cerebral cortex, (I) 200 ´ in subcutaneous nodule. Section (B) is 1000 ´ on a Giemsa stained blood film. All images supplied by D. Milner, Boston.

giostrongylus cantonensis infection or the response of heat shock proteins in Trichinella spiralis infections are immune and/or systemic responses to the organism without its entrance into the CNS [16, 17]. Though these responses may ultimately play no role in the clinical outcome, the neurobiology of this interaction remains fascinating within the context of the BBB. Free-living amoebae represent an interesting subgroup of human parasites because of their tropism for the human CNS. In the case of Entamoeba histolytica, one of the only pathologic Entamoeba spp found in the gastrointestinal tract of hu-

27.2 Malaria: The Plasmodium berghei Mouse Model and the Severe Falciparum Malaria in Man

mans, dissemination is rare to various organs of the body but leads to large necrotic abscess formation, especially in the liver and lungs. Occasionally, E. histolytica will form a cerebral abscess presenting with headache and nonspecific features of encephalitis, meningoencephalitis, or mass lesions. The mechanism by which E. histolytica traverses the BBB is unknown, but the resulting infection is usually fatal. Naegleria fowleri and Acanthamoeba castellanii cause similar diseases resulting in granulomatous encephalitis. A. castellanii usually disseminates to skin and other organs concomitantly with CNS infection. Cytokine priming experiments have revealed that release of endogenous tumor necrosis factor (TNF-a), interleukin-6 (IL-6), and IL-1b are important in defense against A. castellanii infections in mice [18]. The T1 genotype of A. castellanii in an experimental model induces programmed cell death (i.e. apoptosis) in human endothelial cells by specifically activating phosphatidylinositol 3-kinase [19]. The T3, T4, and T11 genotypes bind with higher affinity to human endothelial cells in culture and demonstrate increased cytotoxicity, while the effects of T2 and T7 genotypes on the same human endothelial cell system are significantly less [20]. As A. castellanii represents but one species of the Acanthamoeba spp capable of dissemination within humans, further studies are needed to determine the significance and importance of genotypes and their tropisms. Balamuthia mandrillaris, a causative agent of granulomatous amoebic meningoencephalitis, has been successfully cultured on human endothelial cells from the cerebrospinal fluid of an infected patient and demonstrated cytopathic effects, suggesting important interactions between the amoeba and the endothelial barrier during infection [21]. Phosphatidylinositol 3kinase is also an important mediator of IL-6 release from human endothelial cells exposed to B. mandrillaris, suggesting again that the human response to free-living amoebae is normally preventative of infection [22]. For this reason, immunocompromised patients almost exclusively suffer from disseminated disease. However, the mystery of the CNS tropism of free-living amoebae remains.

27.2 Malaria: The Plasmodium berghei Mouse Model and the Severe Falciparum Malaria in Man (by M.S. Desruisseaux and D. Milner)

Plasmodium spp are parasitic organisms across the animal kingdom, with recent evolutionary studies suggesting that human parasites arose from lateral transfer events from animals or birds to humans. The four human malarias, therefore, are less related to each other than they are to other animal and bird malarias. It is, therefore, not so surprising that P. falciparum infections in humans are very different from the other three species. The life cycle (see Fig. 27.1 A) of these four species involves a mosquito vector transferring infective sporozoites to humans during a blood meal. This is followed by an obligatory liver stage to form the red blood cell-infecting merozoites. Upon release of merozoites from the liver, these parasites begin an asexual reproduction stage with continual production of new merozoites which reinfect red blood cells.

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For three of the species (i.e. P. ovale, P. malariae, P. vivax), this entire life cycle takes place in peripheral blood. In P. falciparum infections, however, the parasitized red cells have the unique ability to cytoadhere to endothelial cells in the post-capillary venules during the latter two-thirds of the life cycle. These late stages are said to be “sequestered”; and they are rarely, if ever, visible in the peripheral blood. The complex mechanisms of cytoadherence include parasite manipulation of the red cell membrane to form knobs (i.e. the expression and insertion into red cell membranes of the parasite proteins PfEMP-1, PfEMP-2, KAHRP, PfHRP1, EDM, RESA), interactions with various receptors on the human endothelial surface of a variety of vascular beds as well as the surface of other cellular fractions such as platelets and cellular microparticles (i.e. CD36, ICAM-1, E-selectin, thrombospondin, chondroition sulfate, GR-1, GAG, fractalkine), and interactions with uninfected red blood cells in the circulation (i.e. rosetting through molecules such as CR-1) [120–123]. There are several in vivo and in vitro examples, however, in which cytoadherence without knob formation occurs, which suggests that sequestration, despite how it is achieved, must be extremely beneficial to the P. falciparum parasite [124–126]. The benefit to the parasite of this ability is probably the capacity to avoid areas of high oxygen tension (e.g. lungs) and areas of immune clearance (e.g. spleen). As sequestration occurs in seemingly every human infection with P. falciparum, the question remains as to exactly what cerebral malaria is and how it comes to manifest in such a small number of patients. Post mortem studies of cerebral malaria in children have demonstrated that fatal events can occur in patients who are diagnosed with clinical cerebral malaria and show the classic pathology of swollen, discolored cerebral cortex, white matter petechial hemorrhages, and the histological correlates of densely packed parasites within endothelial spaces, accompanied variably by fibrin, platelets, white cells, and hemorrhages. However, grossly normal brains with evidence of sequestration only were also seen in a subset of these patients [109]. This finding suggests that the cause of death may be different for these two patterns and thus not wholly tied to a cataclysmic interface with the BBB, but this has yet to be established. Furthermore, it also suggests that in vitro models of disease which focus on the disrupted BBB represent only a subset of patient mortality [110]. Regardless of what is ultimately responsible for human mortality in this infection, studies of the BBB have demonstrated that, at least for the classic pathology as described, there is much interaction and disruption. Though all the features of cerebral malaria cannot be precisely reproduced in the experimental model, there are several parallels in the major pathology of cerebral malaria in the mouse and human models. For example, endothelial cells, which are the mainstay of the BBB, play a vital role in the disease process in both models. It serves as the entry point of the inflammatory cells, and when altered can lead to increased permeability of the BBB [23–25]. Furthermore, certain aspects of the inflammatory response triggered by the malarial proteins are similar in human and mouse cerebral malaria: T-helper cell response, production of interferon gamma (IFN-c), secretion of TNF-a, and monocytic leukocyte cytoadherence to the endothelium [23–25].


P. berghei ANKA strain infections cause a syndrome of severe fatal cerebral malaria in susceptible mouse strains such as CBA or C57BL/6 [23, 25]. This mouse model enables investigators to assess a disease process in which several aspects are analogous to human infection. It is of great importance to note that the P. berghei ANKA strain of malaria on which we will now focus has not been noted to produce red blood cell knobs, as are seen in P. falciparum human infections. Despite this, much of our understanding of the pathogenesis and the immunopathology of cerebral malaria has come from studies of infected rodents. It is well recognized that cerebral symptoms are accompanied by increased permeability in the BBB and cerebral edema in mice infected with P. berghei ANKA [24]. Evidence of leakage of Evans blue stain and iodinated albumin into the brain parenchyma and cerebral spinal fluid (CSF) supports the hypothesis [24]. It is a process that is associated with edema, microhemorrhages, extravasated erythrocytes and monocytes, and endothelial cell damage, as evidenced by electron microscopic studies [24]. Analysis of mouse optic nerves, which are considered equivalent to brain white matter, yields the observation that mild changes in vascular permeability occur very early in the disease process, as early as 2–3 days after P. berghei ANKA infection. These early changes include focal extravasation of horseradish peroxidase alterations in the vascular endothelial junctions and morphological changes in the microglial cells, as evidenced by light and electron microscopy. There is usually a progression to edema and hemorrhage by day 7 of infection [27]. These early changes are postulated to be a result of an interaction between the parasitized red cell and the endothelium, whereas the later changes may be mediated or modulated by the host immune response [27]. Several studies have suggested that a number of processes are involved in the alterations of the endothelial cells of brain capillaries and the ensuing host immune response. These include: (a) sequestration of both infected erythrocytes and monocytes to the capillary beds, (b) direct effects of the parasite particles on the intracellular adhesion molecules, (c) induction of TNF-a either directly or indirectly (T-helper cells stimulate production of IFN-c which in turn induces TNF-a secretion by host monocytes) [25–27].

27.2.1 Upregulation of Intercellular Adhesion Molecules

The intercellular cellular adhesion molecules, such as ICAM-1, are members of the immunoglobulin superfamily. They are expressed constitutively at low levels on the surface of several cell types where they serve as ligands for leukocyte adhesion molecules known as b2 integrins. Their production can be stimulated by a variety of inflammatory cells and parasitized RBCs (pRBCs) via stimuli from chemokines and other proinflammatory molecules [28, 29]. On the endothelial surface of the BBB, they play a central role in the pathogenesis of several processes including cerebral malaria and can mediate the sequestration of leukocytes and platelets [27, 28]. ICAM-1 is thought to be involved in firm adherence of inflam-

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matory cells to the endothelium via its interaction with the integrin lymphocyte function-associated antigen-1 [28]. Lymphocyte adhesion to ICAM-1 is hypothesized to result in the phosphorylation of several cytoskeleton components, thus altering the extracellular matrix and increasing junctional permeability [30]. Once adherent to the endothelium, the inflammatory cells can migrate across and enter into the inflamed tissues [28]. ICAM-1 is also thought to be essential in the transmigration of T-cells into the subendothelial region [28, 30]. It is postulated that the malaria parasite upregulates ICAM-1 expression in part by direct adherence of the infected erythrocytes via specific parasite-derived ligands expressed on the pRBC surface [30]. The alternate pathway through which pRBCs are able to upregulate ICAM-1 is by inducing the secretion of TNF-a, either by direct stimulation or indirectly by the induction of T-helper lymphocyte response, leading to secretion of TNF-a. This cytokine mediates the upregulation of ICAM-1 and other cell adhesion molecules via interactions with the TNF receptor-2 [25, 27, 29, 30]. This interaction is thought to be important, though not entirely necessary, as mice lacking the receptors for TNF-a still exhibit an increase in ICAM-1 expression when infected with the ANKA strain of P. berghei [29].

27.2.2 Secretion of TNF-a

There is extensive evidence indicating that host immune mediators play an important role in the pathogenesis of cerebral malaria in both the mouse and human models [23, 25, 27, 29]. Numerous studies using TNF inhibitors, antibodies, and transgenic mice have demonstrated the importance of TNF-a as a key participant in cerebral malaria [27, 29, 31]. TNF-a is a proinflammatory cytokine produced mainly by activated immune cells. It is produced both as a soluble protein secreted by various inflammatory cells when activated and as a transmembrane protein on the surface of macrophages and activated T-cells [31]. TNF-a promotes extravasation of neutrophils, controls T-cell activation, induces the synthesis of itself and other proinflammatory cytokines, and maintains CNS homeostasis by inducing proliferation of astrocytes and glioma cells. Additionally, TNF-a triggers upregulation of adhesion molecules such as ICAM-1 on the endothelium [29, 31]. This cytokine also causes patchy demyelination and axonal damage in the CNS [26]. The causes of TNF-a release appear to be multifactorial. Malarial toxins are able to directly induce host monocytes to secrete TNF-a by an unclear mechanism that seems to be protein kinase-dependent [25, 31]. T-helper lymphocyte activation by the parasite antigens and the ensuing production of IFN-c leading to the secretion of TNF-a may also be required for the development of cerebral malaria, since athymic mice as well as mice treated with anti-CD4 antibody seem to be protected from cerebral malaria [23, 25, 27]. Brain, astrocytes, microglia, and neurons are able to produce TNF-a locally after challenge with various stimuli [31]. TNF-a binds with equal affinity to two distinct receptors: TNF receptor-1 and TNF receptor-2. Both receptors are expressed in equal amounts by most cells. Usually,


receptor-1 mediates cytotoxic activity while receptor-2 is responsible for proliferative activities [31]. In mouse cerebral malaria, it has been determined that TNF-a exerts its actions mainly through TNF receptor-2. This receptor preferentially interacts with the membrane-bound TNF rather than the soluble form [27, 29]. During infection with P. berghei ANKA, TNF receptor-2 has been shown to be absolutely necessary for the development of cerebral malaria and for death due to cerebral malaria [27, 29]. Receptor-2 null mice (TNFR2–/–) do not become sick or die during acute infection as compared with wild-type mice or tumor necrosis factor receptor-1 null (TNF1–/–) mice, despite identical levels of parasitemia [27, 29]. Rather, they die 3–4 weeks post-infection due to a high parasite load and anemia [27]. However, though receptor-2 is thought to offer some degree of protection against vascular permeability in the brain as evidenced by a decrease in the leakage of Evans blue material in the brain of TNFR2–/– mice [27, 29], this reduction is shown to be both receptor-1 and receptor-2 mediated as TNFR1–/– mice also show attenuation of permeability [29]. Moreover, both receptors seem to be similarly influential in leukocyte sequestration and indirectly, endothelial cell damage. Neither TNFR2–/– nor TNF1–/– mice demonstrate significant cytopathic endothelial cell alteration upon evaluation of the cortical capillaries of P. berghei ANKA-infected mice or in vitro on brain mouse vascular endothelial cells [27, 29]. The specific effect of receptor-2 on ICAM-1 upregulation seems to be controversial, as some studies emphasize the importance of that receptor in ICAM-1 expression [27] and some studies show a lack of correlation [29]. Lymphotoxin-a, formerly TNF-b, a member of the tumor necrosis factor family, may be equally important in the development of cerebral malaria. Lymphotoxin-a is also able to bind both tumor necrosis factor receptors-1 and -2, and may be able to upregulate the adhesion molecules as well. Mice deficient in lymphotoxin-a seem to be protected from developing cerebral malaria [23, 31].

27.2.3 Microglial Activation

Microglia are cells of macrophage/monocyte origin and a part of the local immune response in the central nervous system. When activated by different pathogenic stimuli, they respond in various ways including morphological alterations. As mentioned previously, they can be stimulated by IFN-c to locally secrete cytokines such as TNF-a, IL-1b, and IL-6 in the CNS as well as reactive oxygen radicals [32, 33]. When activated, the processes on the microglia become less ramified and take on a more amoeboid shape, i.e. more macrophage-like [33, 34]. This is thought to be a reparative effort as a result of compromise of the BBB, an indication of protein phagocytosis [26]. These changes are evident as early as 3 days post-infection, increasing in severity with the progression of disease [33]. The distribution of the microglia is also altered: instead of an even distribution within the parenchyma, the microglia seem to be more concentrated in close proximity to the blood vessels [33].

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In addition to activated microglia cells contributing to the immunopathology, there is evidence that extravasated monocytes in the perivascular regions are also a major factor in local immune response of the brain [33].

27.2.4 Vascular Damage

Two studies have explored the role of magnetic resonance imaging (MRI) as a noninvasive method of following the evolution of cerebral malaria in P. berghei ANKAinfected mice. These studies demonstrated a marked disruption of cerebral blood flow in several areas of the brain during malarial infection [34, 35]. With angiograms, only the larger blood vessels such as the carotid arteries and the Circle of Willis are clearly visualized [35]. Parenchymal lesions indicative of blood brain barrier disruption are also observed with MRI, as are regions of hemorrhage; confirming the histological findings [35]. These studies also identify N-acetyl aspartate as a biological marker for disease progression in cerebral malaria [34, 35]. N-acetyl aspartate is an acetyl group donor produced mainly in neuronal mitochondria. It is necessary for myelination. A decrease in N-acetyl aspartate levels is usually an indication of neuronal damage [34, 35]. With cerebral malaria, there is clearly an inverse relationship between disease severity and N-acetyl aspartate levels in the brain. In addition to the few reports of imaging studies on humans showing white matter changes, cortical infarcts, and edema, this information may be very helpful in providing a noninvasive method to monitor the progression of disease and the degree of damage that ensues with cerebral malaria [34]. Recent data from our laboratory demonstrate that there is an increase in the expression of endothelin-1, a powerful vasoconstrictor, in the brains of P. berghei ANKA-infected mice [36], which may in part explain the reduction in cerebral blood flow observed in these mice [36]. In summary, cerebral malaria is an entity in which there is a strong correlation between evidence of destruction of the BBB and the development of symptoms. Damage to the endothelial cells is observed very early post-infection, even prior to the onset of symptoms, as demonstrated by leakage of horseradish peroxidase in the brain parenchyma as well as subtle transformation of the microglia [26]. These early alterations are possibly mediated by direct parasite factors that either act directly on the endothelium by inducing ICAM expression or by triggering the secretion of TNF-a by activated monocytes and microglia [26–28, 30]. As the disease progresses and symptoms become evident, these changes in the endothelium become more apparent and more diffuse. This is associated with hemorrhage, albumin leakage and edema, extravasation of erythrocytes and monocytes, and marked changes in the morphology of the microglia [25, 26, 32–34]. It is now widely accepted that this process is triggered by activation of the host immune system and that TNF-a promotes upregulation of cell adhesion molecules and induces changes in the endothelium that compromise permeability [23, 25, 27, 29, 31]. There is also evidence that T-helper lymphocyte activation may also be im-

27.3 Trypanosomiasis: African and American Parasites of Two Distinct Flavors

portant, since asplenic mice and mice treated with anti T-cell antibodies are protected from the development of cerebral malaria even when treatment is initiated late in the course of the disease, presumably in part because of some degree of reversal of the permeability in the BBB [37]. This interplay between the cytokines, chemokines, and the BBB in mouse models is consistent with findings from post mortem studies in humans with cerebral malaria.

27.3 Trypanosomiasis: African and American Parasites of Two Distinct Flavors (by H. Tanowitz, M. S. Desruisseaux, and A. Mott)

Both African and American trypanosomiases have been known to cause CNS injury. However, there are major differences in the manifestations and duration of the disease between the different species, and even among various strains within the same species. African trypanosomiasis or sleeping sickness occurs after infection with either Trypanosoma brucei gambiense, which causes chronic sleeping sickness or T.b. rhodesiense, which causes acute sleeping sickness in endemic regions of sub-Saharan Africa [38–41], whereas American trypanosomiasis or Chagas’ disease is caused by T. cruzi, a hemoflagellate protozoan endemic in Mexico, Central and South America [42–44]. Save for duration and acuity, the symptoms of sleeping sickness are essentially the same for both the acute and the chronic forms of African trypanosomiasis. They are characterized by a hemolymphatic stage in which the most common complaints are fever, headache and arthralgias, and muscle aches. The symptoms are sometimes so minor that patients may not feel the need to seek medical advice [38, 39]. This is accompanied by lymphadenopathy, hepatosplenomegaly, anemia, ascites and edema, among other signs [38–40]. The hemolymphatic period is followed by a meningoencephalitis stage characterized by sleep disturbances, altered mental status (i.e. psychosis, personality disorders), abnormal reflexes, convulsions, hemiplegia, worsening consciousness, coma, and eventually death if left untreated [38–41, 45]. This phase is characterized by alterations in the regulation of several pro- and anti-inflammatory cytokines both systemically and in the CNS, as well as marked changes in nitric oxide synthesis [38–41, 45, 46]. In addition, astrogliosis and mononuclear infiltrates in the white and grey matters are observed during this process, even prior to any other brain lesions, correlating well with the changes in the sleep cycle observed with the disease [39, 40, 46]. There is ample data demonstrating the presence of T. brucei in the brain during infection, both in early and late stages [47–49] and one of the diagnostic criteria for sleeping sickness is demonstration of parasites in the cerebral spinal fluid [38, 41, 50]. There is also evidence for obvious breakdown of the BBB, especially in the late stages of disease, with edema and elevated brain water content, as well as leakage of rhodamine dye into the brain parenchyma throughout the grey and white matter of the brain [46, 47]. However, despite a plethora of

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investigative efforts, the exact mechanisms of these processes have never been clearly elucidated. Several authors have tried to clarify the progression of neuropathology that occurs in African trypanosomiasis.

27.3.1 CNS Pathology in African Trypanosomiasis

Early parasitic infiltration of the brain in animal models occurs in areas where there is paucity in the BBB development. Early damage is thought to occur in the choroid plexus and the periventricular regions, with parasites penetrating these tissues via some unclear mechanism and spilling into the cerebral spinal fluid [39, 40, 46, 47]. This results in chronic meningitis whereby inflammatory cells and Mott cells, plasma cells containing immunoglobulin, are able to permeate throughout the meninges [38, 39, 45, 46]. Access to the perivascular space is then afforded to the parasite and the inflammatory cells by direct extension, leading to the formation of perivascular cuffing [39, 45, 51]. One of the hallmarks of sleeping sickness is autoantibody production. Antibodies against erythrocytes, muscle cells, and single-stranded DNA, as well as against neurons, gangliosides, and galactocerebrosides have been observed [39, 46]. There are several implications to this, including demyelination observed in late stages of the disease, tissue damage and hemolytic anemia among others. The autoantibody production might be important in the autonomic dysfunction as well as the myocarditis observed in the disease [45].

27.3.2 Cytokines and Endothelial Cell Activation

In the presence of inflammatory stimuli activated endothelial cells express cellular adhesion molecules which allow sequestration and transmigration of leukocytes across the endothelium. In vitro studies have demonstrated that trypanosome extracts, as well as the trypanosomal variable surface glycoproteins (VSGs) are able to significantly induce a transient increase in the expression of ICAM1, VCAM-1, and E-selectin on the surface of human bone marrow endothelial cells, comparable to levels obtained upon stimulation with TNF-a [52]. These molecules are able to mediate changes in the extracellular matrix and the cytoskeleton of the endothelium, which may lead to increased permeability [52]. T. brucei mediates the activation of endothelial cells by inducing an increase in cytokines such as TNF-a and IFN-c. Several studies demonstrate that cytokines are markedly elevated in infected subjects at all stages of infection, and that their levels continue to increase as the disease progresses [53]. Plasma levels of IFN-c increase as early as day 7 post-infection. The levels of IFN-c and TNF-a in the brain increase exponentially with the severity of the neuropathology [53]. A similar relationship exists with


IL-1b and brain pathology, though the correlation is not as strong as with TNFa and IFN-c [53, 54]. These cytokines may act synergistically in altering the immune response and in modulating the expression of cell adhesion molecules [55]. In addition, when exposed to endothelial cells, they may be implicated in promoting increased permeability [40]. However, a negative relationship exists between the anti-inflammatory cytokines, such as IL-10 and IL-6, and brain pathology. The levels of these factors are elevated at stages when brain pathology is mild in infected animals [53]. It should be noted that, while the CNS levels of IFN-c increase with disease progression, the plasma levels of the cytokine decrease. The same pattern is observed with systemic TNF-a, where concentrations decrease in late disease, suggesting that the CNS concentration of these cytokines originates from an endogenous source [53]. It is postulated that an early increase of systemic IFN-c and other cytokines is responsible for astrocyte and microglial activation in the brain. These cells in themselves are able to produce inflammatory cytokines; and this may provide an explanation for the endogenous production of cytokines in the brain [53].

27.3.3 Astrocytosis and Microglial Cell Activation

Astrocyte activation is early evidence of CNS histopathology in sleeping sickness, with morphological changes evident even prior to any major brain lesions [39, 56]. Early in the disease process, activation occurs in a diffuse manner. However, as the infection progresses, the distribution of activated clusters of microglia is more prevalent in regions like the hypothalamus, the cerebral cortex, and the septum [56, 57]. There is a marked hypertrophy in these cells at later stages of disease; and these changes appear to correlate with the changes in sleep cycle characteristic of sleeping sickness [56, 57]. Microglia and astrocytes express certain chemokines involved in recruitment of systemic T lymphocytes into the CNS. Notably, the parasites are able to stimulate CD8+ T lymphocytes to secrete IFN-c, a cytokine known to promote parasite growth and proliferation [56]. Trypanosomal factors are also able to mediate inducible nitric oxide synthase production within astrocytes and microglia in the brain [39, 56].

27.3.3.1 Nitric Oxide Nitric oxide (NO) under physiological conditions causes smooth muscle cell relaxation and inhibits platelet aggregation, thus ensuring adequate blood supply to various organs. Excessive NO production may be important in vascular permeability and may contribute to BBB breakdown and the transmigration of inflammatory cells [46, 51, 58]. Under physiologic situations, NO is constitutively synthesized by nitric oxide synthase in endothelial cells (eNOS or NOS3) and neuronal cells (BNOS or NOS1) and is dependent upon calcium-calmodulin interactions

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[51]. In inflammatory settings, in the presence of certain cytokines and microorganisms, the production of NO is increased via activation of inducible nitric oxide synthase (iNOS or NOS2) in macrophages and brain microglia in a process that is independent of calcium [51]. It is believed that there is a dual action exerted by NO. The constitutively synthesized NO, when found in excess, has a cytotoxic role and is speculated to have some effect in the permeabilization of the BBB with trypanosomal infection. The concentration of constitutive NOS is found to be directly correlated with the evolution of disease [51]. However, when surplus NO is synthesized in the macrophages, there appears to be an attenuation of T-helper cell response and immunosuppression which aids in the survival of the parasite [58]. Though the specific role of NO and its toxic effects on CNS events in trypanosomal infection remains to be elucidated, in vitro studies using purified mouse brain astrocytes and microglia demonstrate an increase of iNOS when these cells are cocultured with trypanosomes, leading to increased NO release. This implies that there is direct contact of these cells with trypanosomal components in the brain of infected subjects [59]. Indeed, in mice infected with T. b. brucei, increased iNOS activity can be observed in mononuclear cells in the meninges, the choroid plexus, and at times in the parenchyma [60]. The presence of iNOS in these regions appears to precede neurological signs [60]. IFN-c is also able to induce iNOS production in those cells and acts synergistically with trypanosomes to induce these changes [59].

27.3.4 American Trypanosomiasis (Chagas’ Disease)

Chagas’ disease is most often associated with acute myocarditis and chronic dilated cardiomyopathy, as well as the mega syndromes. Although the brain is involved in T. cruzi infection, it has not been extensively investigated. The CNS is involved in congenital Chagas’ disease which is most prevalent in highly endemic areas. Neonates may suffer from seizures, jaundice, and be clinically indistinguishable from the manifestations of congenital toxoplasmosis or other etiologies in the TORCH syndromes. The acute infection phase occurs in approximately 1% of cases and occurs mostly in young children, less than 2 years old [29, 43, 52]. It is characterized by parasitic penetration into the CNS, with resultant inflammatory foci of glial nodules. Trypomastigotes at that stage can sometimes be present in the cerebral spinal fluid; and amastigotes can be found in glial cells and macrophages [39, 43]. The parasites are postulated to enter the brain parenchyma through the pial vessels; and invasion of the CNS may occur because of immaturity of the BBB in that age group [43]. It is important to note that acute T. cruzi infection is associated with increased levels of TNF-a which can modulate alterations in the BBB [42, 43]. There is then an intermediate phase in the majority of patients where there may be waning of the inflammation [39]. This is followed by the chronic stage which may occur years after infection. This includes a wide array


of CNS abnormalities, including damage to the peripheral autonomic and skeletal muscle innervations via some autoimmune mechanism [39]. More recently, Chagas’ meningoencephalitis has been observed in the setting of HIV/AIDS, where the clinical and radiographic manifestations are indistinguishable from toxoplasmosis in the same setting [61]. The involvement of the CNS is rare, though the presence of the parasite in the CNS and especially the CSF during the acute phase of the disease has been demonstrated [111–114]. The first report of reactivation in an AIDS patient was in 1990 [115] and there have been several reports since that time [116–119]. In patients with advanced AIDS, cerebral infection is the prevalent form of reactivation disease. The clinical features of these infections are similar to those of CNS toxoplasmosis. The CNS disease normally presents as multifocal, necrotizing encephalitis [113, 115, 118]. In some cases, the trypomastigote form of the parasite can be visualized in CSF, but generally the diagnosis is confirmed by intracranial biopsy. The amastigote form of the parasite can be observed in many cell types of the brain tissue by light or electron microscopy [111]. At this time, no efficacious treatment protocol has been defined and the number of case fatalities is high, with a short duration of survival. An acute CNS reactivation of chronic Chagasic disease may also occur in immunosuppressed patients, such as individuals with leukemia and post-transplantation [39, 44]. In these patients, there is clear evidence of encephalitis and inflammatory foci with multiple amastigotes [39, 44]. Imaging of the brain is significant for the presence of ring-enhancing lesions in the white matter, subcortical regions, and the cerebellum, with very little edema [44]. Necrosis and hemorrhage is apparent both histologically and by MRI [39, 44]. Trypomastigotes of T. cruzi invade endothelial cells, resulting in an increased synthesis of cytokines such as TNF-a and IL-1b, and in addition, an increased synthesis of the vasoactive peptide, endothelin-1 [62, 63]. These findings could be responsible in part for vascular spasm and loss of the integrity of the BBB. Moreover, T. cruzi infection of endothelial cells have been reported to alter gap junctions between endothelial cells which also lead to alterations in the BBB [64]. Finally, recent evidence indicates that, like African trypanosomiasis, T. cruzi infection causes an increase in inflammatory mediators in the brain [65]. Trypanosomal disease of the CNS involves a complex process in which the details are not altogether clear. There is evidence of direct trypanosomal penetration in the CNS in both African and American disease, mostly in areas of decreased BBB stability. However, as disease progresses, the distribution of parasites in the brain parenchyma may not follow a specific pattern. There is a surge of proinflammatory cytokines with infection, which can help to stimulate certain adhesion molecules on the surface of endothelial cells, thereby bringing about compromise in the BBB permeability. There is also an increase in the synthesis of NO, which can bring about cell damage and can lead to increased permeability of the endothelium. There is demonstration of glial cell activation, and in the case of T. cruzi, there are trypanosomal forms within these glial cells. There is also an autoimmune phenomenon in which anti-host cell antibodies are synthesized, causing a lot of destruction to many structures inside and out

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of the CNS. Moreover, at the later stages of disease, there is evidence of hemorrhage (T. cruzi) and edema (T. brucei), which further strengthens the hypothesis that there is widespread compromise of the BBB in these disease entities.

27.4 Toxoplasmosis: Transgression, Quiescence, and Destructive Infections (by L. Weiss)

Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that can infect any mammal. Infection results from ingestion of either the tissue cyst stage or oocysts. Initial infection in the gastrointestinal tract is followed by dissemination of tachyzoites and acute disease, which is often asymptomatic and self-limiting. Studies in mice and rats after oral infection with oocysts suggest that T. gondii first infects mesenteric lymph nodes, then the spleen, and subsequently the brain, heart, and other organs via the bloodstream [66]. Recent observations in mice suggest that T. gondii may transverse the BBB inside CD11c and CD11b expressing leukocytes, prior to replication in the brain [67]. Other studies have demonstrated that T. gondii microneme protein 2 (MIC2) can bind to ICAM-1 and that, following this, the organism actively penetrates endothelial cell monolayers [68]. Interestingly, the integrity of the endothelial cell barrier was not altered during parasite transmigration. ICAM-1 is upregulated during the course of T. gondii infection [68]. Following this acute dissemination, a latent phase develops with tissue cysts containing bradyzoites being present in brain, muscle, and other tissues, which lasts for the lifetime of the host. Tissue cysts persist within astrocytes and neurons of the brain and elicit little to no inflammatory reaction [69]. Tissue cyst rupture and bradyzoite differentiation to the tachyzoite stage are thought to occur intermittently, but disease is limited due to the immune response of the host [70, 71]. T. gondii is an important opportunistic infection of the CNS in AIDS patients as well as other immunocompromised patients, such as transplant recipients and those receiving chemotherapy. In the setting of immune suppression, encephalitis often results from a reactivation of the tissue cyst stage in the brain [72]. In these immunocompromised hosts when tissue cysts rupture, tachyzoites can replicate freely in the brain, resulting in lysis of their host cells and in encephalitis with necrotic foci in the brain. IFN-c is the primary cytokine controlling both the acute and chronic stages of infection [73–75]. Elevated levels of IFN-c and the cytokines IL-1b and TNF-a are present in the brains of T. gondii infected mice [76–78], and the administration of anti-IFN-c results in an increase in the amount of encephalitis in infected mice [79, 80]. Protection against both the acute and chronic stages of T. gondii requires cells from both the hematopoietic and nonhematopoietic lineages [81]. The effector cell populations in the brain include CD4+ and CD8+ T cells, microglia, astrocytes, dendritic cells, B cells and an IFN-c producing a non-T cell population [82, 83]. Neurons, astrocytes, and microglia have all been shown to produce IFN-c in

27.4 Toxoplasmosis: Transgression, Quiescence, and Destructive Infections

vitro and these cells may be important sources of IFN-c [84, 85]. IFN-c stimulates the microglia to produce TNF-a which upregulates ICAM-1 [86]. Neutrophils are the first cells to be recruited to sites of infection and they play a critical role in retarding intracellular parasite growth and lysing extracellular parasites [87]. In mouse models of toxoplasmosis, depletion of neutrophils before infection leads to an increase in parasite load and an impaired ability of the host to produce IFN-c, TNF-a and IL-12 [88]. Likewise, mice in which neutrophils had impaired migration had increased parasite loads and decreased levels of IFN-c [89, 90]. Dendritic cells serve as antigen-presenting cells (APC) early in infection and produce IL-12, priming the differentiation of CD4+ T cells to produce IFN-c and thus ensuring an adaptive immune response [91]. Infected astrocytes produce factors that enable dendritic cells to differentiate into APC and thus help initiate the intracerebral immune response. Brain dendritic cells represent a novel type of APC, because both astrocytes and microglia (the other known APCs) require IFNc, whereas brain dendritic cells do not. This feature may explain how intracerebral T cell activation occurs. IL-12 in conjunction with TNF-a, IL-1b and IL-15 also activates natural killer (NK) cells to produce IFN-c [92–94], priming macrophages to produce IL-12, thereby amplifying local IFN-c production [95] with IFN-c and IL12, driving a T cell Th1 response [92]. Studies indicate the major sources of IFN-c are CD4+ and CD8+ T cells that are recruited into the brain during infection [96, 97]. CD8+ T cells are thought to be the primary effector cell against T. gondii in the brain, with CD4+ T cells playing a synergistic role [72, 92, 98, 99]. CD8+ T cells exhibit cytotoxic lymphocyte (CTL) activity in a MHC-restricted manner [100–103]. In the chronic phase of infection, prevention of TE depends upon CD8+ T cells and IFN-c [96]. During the chronic stage of the infection, the Th1 cytokines IL1, IL6, TNF-a, and IFN-c decrease, while IL-10 increases in the brain [96]. Microglia, the resident macrophage population in the brain, are probably a major effector cell in the prevention of T. gondii tachyzoite proliferation in the brain [86, 104, 105]. Astrocytes can also inhibit growth of T. gondii in the brain [106] and produce proinflammatory cytokines [107]. These, together with the cytokines produced by activated microglia, probably play an important role in inducing the infiltration of immune cells into the brain. The proinflammatory byproducts of activation such as NO, while inhibiting parasite replication in the brain, are themselves detrimental to neurons and may also contribute to defects in the BBB. T. gondii can infect microvascular endothelial cells and IFN-c has been demonstrated to inhibit T. gondii replication in these cells [108]. Since one of the first steps in the development of cerebral toxoplasmosis is penetration of the BBB, IFN-c-induced inhibition in these cells may be important for limiting the replication of T. gondii in the brain.

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27.5 Conclusion

Human parasitic infections remain crucially important within the scope of human disease. The very complex interactions of the BBB with a subset of these parasites seem to lead to the greatest morbidity and highest mortality in each disease group. Further understanding of these relatively rare events within the context of a global understanding of the diseases as a whole is a vital aspect of reducing disease mortality in human populations – especially for those who suffer most. Their suffering is not due to ignorance, but is a reflection of the larger socioeconomic or political circumstance.

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28 The Blood Retinal Interface: Similarities and Contrasts with the Blood-Brain Interface Tailoi Chan-Ling

28.1 Introduction

The mammalian retina forms as an outgrowth of the diencephalon during early embryonic development. Embryologically, the retina is thus part of the central nervous system (CNS) and its blood vessels share many characteristics with those of the brain, including barrier properties (Bill et al. 1980), the ability to autoregulate (Bill and Nilsson 1982, 1985; Bill and Sperber 1990), close contact with astrocytes (Ling and Stone 1988; Ling et al. 1989; Chu et al. 2001; ChanLing et al. 2004 a, b), association with microglia (Hollander et al. 1991), pericyte ensheathment (Chan-Ling et al. 2004b; Hughes and Chan-Ling 2004), and involvement in immune responses (Chan-Ling et al. 1992; Hu et al. 1998 a, b). However, whereas the brain is encased in the cranium with pial coverage and is bathed in cerebrospinal fluid, the retina is located inside the eyecup and its microvasculature is adjacent to and adjoins nonCNS vascular beds. These junctions between vasculatures with CNS and peripheral characteristics in the eye are unique in their cell biology, are inherently susceptible to breakdown of the blood-retinal barrier (BRB), and play a prominent role in immune activation. The transparency of the ocular media makes it possible to view the retinal blood vessels in situ, in turn enabling the detection of cardiovascular pathology such as nipping of arterioles caused by high blood pressure and changes in arteriole and venular walls due to atherosclerosis or diabetic retinopathy. In contrast to the brain, the anterior location of the globe also renders it readily accessible to experimental manipulation, such as intraocular injection. For these reasons, the BRB has often been studied as a model to gain insight into the structure of the blood-brain barrier (BBB) as well as into various CNS disease processes. However, it is necessary to gain a full understanding of the unique features of the various blood-retinal interfaces before attempting to use the retina as an experimental model of the CNS. Changes in the BRB are of great importance in their own right, given that breakdown of this barrier is an inherent component of the pathogenesis of most sight-threatening retinopathies. This Blood-Brain Interfaces: From Ontogeny to Artificial Barriers. Edited by R. Dermietzel, D. C. Spray, M. Nedergaard Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31088-6

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28 The Blood Retinal Interface: Similarities and Contrasts with the Blood-Brain Interface

chapter reviews our current understanding of blood-retinal interfaces, highlighting both unique features and those shared with other CNS vessels.

28.2 The Inner and Outer BRB

The BRB is essential for control of the microenvironment of the retinal parenchyma, given that it physically impedes the diffusion of substances between the blood and nervous tissue. In humans and several other mammalian species, the BRB is composed of two parts (Fig. 28.1 A) because the retina has a dual vascular supply, the inner two-thirds of the retina being nourished by intraretinal vessels and the outer one-third being supplied by choroidal vessels. The barrier properties of the inner retina depend on the tight junctions between adjacent endothelial cells of the intraretinal capillaries and other vessels (Fig. 28.1 B, H), whereas those of the outer retina depend on the tight junctions between adjacent cuboidal cells of the retinal pigment epithelium (RPE; Fig. 28.1 C, D). These two barriers have been described as the inner and outer BRB, respectively (Cunha-Vaz 1976). The inner BRB separates the retina from circulating blood, whereas the outer BRB, which is formed by the single layer of cuboidal cells that constitute the RPE, separates the neural retina from the fenestrated capillaries of the choroids (Tyler and Burns 1991). The capillaries of the choroid are highly permeable and manifest only a low level of expression of the tight junction protein occludin between adjacent vascular endothelial cells (Flage 1977; Morcos et al. 1999) (Fig. 28.1 E). The vessels of the retinal circulation derive from mesenchymal tissue that enters the eyecup through the optic fissure and spreads through the superficial layers of the retina (Ashton 1970; Chan-Ling et al. 1990). The vessels therefore form initially at the inner surface of the retina, with branches subsequently extending from these superficial vessels into the deeper layers (Michaelson 1954). The retinal vasculature resembles the cerebral circulation in terms of vessel structure and permeability (Bill et al. 1980), flow rate, oxygen clearance (with the venous partial pressure of oxygen typically being 60–65% of the arterial value; Bill and Nilsson 1982), and the ability to autoregulate flow in response to changes in arterial pressure and oxygen tension (Alm and Bill 1973; Bill and Nilsson 1982).

28.3 The Choroidal Vasculature

The choroidal vasculature lies outside the CNS, supplying the retina by diffusion through Bruch’s membrane and the RPE. The outer region of the retina is the only part of the CNS of an adult mammal that is supplied via diffusion from a distance by extrinsic vessels, rather than by intrinsic vessels. The choroidal vascular plexus thus differs from the cerebral and retinal circulations and its vessels are distinct from CNS vessels in terms of their location, structure (lacking glial ensheath-

28.5 Ensheathment and Induction of the Inner BRB by Astrocytes and Müller Glia

ment), flow rate, permeability, and oxygen clearance (Bill et al. 1983). Direct studies of blood flow have shown that the flow rate in the choroidal circulation does not decrease during hyperoxia or increase during hypoxia, but rather varies linearly with vascular resistance (Bill and Nilsson 1982). These observations suggest that the choroidal circulation, unlike the CNS circulation, does not autoregulate its flow. However, more recent studies based on fluorescein angiography (Flower 1990; Flower and Klein 1990) suggest that choroidal blood flow does indeed decrease during hyperoxia. Furthermore, the pH of the subretinal space was found to change in response to an increase in intraocular pressure (Yamamoto and Steinberg 1992), suggesting that the choroidal circulation may be able to regulate its flow, albeit to a small extent and only in the vicinity of the optic disk. Not only do choroidal capillaries lack CNS-like barrier properties, as indicated by the extravasation of intravascularly introduced horseradish peroxidase (HRP) into the parenchyma surrounding these vessels (Morcos et al. 1999), but they are fenestrated and highly permeable. Their permeability to plasma proteins is five times that of the capillaries of the kidney, which are also fenestrated, and 10–30 times that of the capillaries of striated muscle, which are not fenestrated (Bill et al. 1980). The permeability of choroidal capillaries to smaller substances, such as sodium ions, is about 30 times and 50 times that of the capillaries in heart and skeletal muscle, respectively. The tight junctions between adjacent cells of the RPE thus separate the retina from the leaky blood vessels of the choroid.

28.4 Characteristics of Intraretinal Blood Vessels

The intraretinal vasculature consists largely of two layers, the inner or superficial retinal plexus, and the outer or deep retinal plexus. The superficial plexus is located in the ganglion cell and nerve fiber layers; and the deep plexus is located at the junction between the inner nuclear and outer plexiform layers (Fig. 28.1 A). The superficial retinal plexus contains vessels of different calibers, including arterioles, venules, capillaries, and postcapillary venules, whereas the deep plexus consists mostly of capillary-sized vessels (Fig. 28.1 A). The deep retinal plexus has been described as being more venous in nature, with postcapillary venules also being evident in this plexus.


Although the precise nature of the interaction between blood vessels and surrounding astrocytes in the CNS is not completely understood, in vitro evidence suggests that astrocytes or substances released by them contribute directly to the formation and maintenance of functional barrier properties (Tao-Cheng et al. 1987, 1988; Laterra et al. 1990; Tontsch and Bauer 1991; Raub et al. 1992;

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Fig. 28.1 (A) The intraretinal plexus is found predominantly in two layers, the inner or more commonly known as the superficial plexus, and the outer or deep retinal plexus. The superficial plexus is located in the ganglion cell and nerve fibre layers and the deep retinal plexus is located at the junction between the inner nuclear and outer plexiform layers. The outer one-third of the retina is nourished via diffusion from the choroidal vessels. The outer blood retinal barrier is formed by the simple layer of

cuboidal cells that constitute the retinal pigment epithelium (RPE). The RPE separates the neural retina from the fenestrated capillaries of the choroid. (B–E) Light microscopic analysis of the expression of the tight junction proteins, occludin and claudin-1, in the rabbit eye (Morcos et al. 2001). (B) Expression of occludin in the retinal blood vessels within the myelinated region of the adult rabbit retina. The continuous expression of the protein was evident at the


Wolburg et al. 1994; Gardner et al. 1997). Studies with an animal model of retinopathy of prematurity have revealed a relation between breakdown of the BRB and degeneration of astrocytes (Chan-Ling and Stone 1992; Chan-Ling et al. 1992). Furthermore, astrocytes express occludin in vitro (Bauer et al. 1999). The level of occludin expression in retinal endothelial cells decreases in animals with experimental diabetes at the same time as retinal permeability increases (Antonetti et al. 1998). Moreover, occludin expression decreases together with that of zonula occludens-1 (ZO-1), a peripheral membrane protein associated with tight junctions, in the cerebral vascular endothelium during neutrophil-induced breakdown of the BBB (Bolton et al. 1998). Since the first demonstration that the barrier properties of blood vessels are influenced by the vessel environment (Stewart and Wiley 1981), evidence has accumulated to suggest that astrocytes induce endothelial cells to develop barrier properties, specifically to form complex tight junctions with their neighbors and to express various barrier-specific enzymes and molecular transporters (Beck et al. 1984, 1986; Stewart and Coomber 1986; Maxwell et al. 1987; Stewart and Hayakawa 1987; Tao-Cheng et al. 1987; Brightman 1989; Dehouck et al. 1990; Hollander et al. 1991; Tontsch and Bauer 1991). Iris vessels have been shown to become impermeable on association with implanted astrocytes (Olson et al. 1983; Janzer and Raff 1987; Tout et al. 1993). The arteries of the retina, iris, and ciliary processes are all branches of the ophthalmic artery. However, ocular ves-

Fig. 28.1 (continued) interfaces between adjacent endothelial cells of vessels in the myelinated streak supportive of their tight barrier properties, demonstrated using intravascular injection of the tracer horse radish peroxidase. (C) Expression of the tight junction protein, occludin, in the RPE of the rabbit eye. Occludin was detected around the entire circumference of RPE cells. (D) Expression of the tight junction protein, claudin-1, in the RPE of the rabbit eye. Claudin-1 was detected around the entire circumference of RPE cells. (E) Expression of occludin in vessels of the choroid of the rabbit eye. The choroidal vessels exhibited only weak and irregularly distributed occludin immunoreactivity, supportive of the leakiness of choroidal blood vessels. Occludin was not evident at most endothelial cell junctions in the choroidal vascular plexus, in marked contrast to the patterns of expression apparent in vessels with barrier properties.

(F) Relation of astrocytes to blood vessels in the superficial retinal plexus shown with an antibody to GFAP conjugated to Texas red and Griffonia simplicifolia isolectin B4 conjugated to FITC. A vein runs obliquely across this field and is wrapped by many astrocyte processes. (G) Muller cells forming the glia limitans of the deep retinal vessels in adult cat retina, shown by immunohistochemistry. This field shows the broken edge of a wholemount preparation, labelled with 4D6, a Müller cellspecific antibody. The inner endfeet of Müller cells are brightly fluorescent, at top. Their radial processes can be traced across the inner plexiform and nuclear layers. At the outer margin of the inner nuclear layer, they outline a capillary of the outer layer of retinal vasculature. (H) Vessels at the edge of the region vascularized in a normal P12 retina. Blood cells and the HRP are trapped within the vessel lumen; and the spaces between vessels appear free of peroxidase.

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sels vary from being highly permeable in the choroid, ciliary processes, and meningeal aggregates, to less permeable in the iris, to impermeable in the retina. Müller cells are the radial glia of the retina, with the inner and outer endfeet of these cells expanding to form the external and internal limiting membranes of this tissue. Glial ensheathment of the superficial retinal vascular plexus is mediated by astrocytes, Müller cells, and, to a much lesser extent, perivascular microglia (Bussow 1980; Stone and Dreher 1987; Schnitzer 1988; Ling et al. 1989; Hollander et al. 1991) (Fig. 28.1 F), whereas vessels of the outer plexus are ensheathed by Müller cells only (Kondo et al. 1984; Hollander et al. 1991) (Fig. 28.1 G). Despite the difference in glial ensheathment between these two vascular beds, occludin expression is evident in both the inner and outer plexuses of the adult rat retina (Chan-Ling et al. 2005). These observations led us (Tout et al. 1993) to suggest that Müller cells share the ability of astrocytes (Tao-Cheng et al. 1987; Bouchard et al. 1989; Brightman 1989; Dehouck et al. 1990) to induce the development of barrier properties in endothelial cells. Implantation of cultured Müller cells into the anterior chamber of the rat eye revealed that the cells formed aggregates and became associated with iris vessels (Olson et al. 1983; Janzer and Raff 1987; Tout et al. 1993). Assessment of the barrier properties of the vessels associated with the cell aggregates by intravascular perfusion either with Evans blue to visualize serum albumin by light microscopy or with HRP to detect vessel leakage by electron microscopy showed that Müller cells indeed share with astrocytes the ability to induce the development of barrier properties in endothelial cells (Tout et al. 1993). Müller cells thus appear to induce the formation of tight junctions.

28.6 BRB Properties of Newly Formed Vessels

Astrocytes are intimately associated with the vasculature during development of the mammalian retina (Chan-Ling 1994). Both endothelial cells and astrocytes proliferate during retinal vascular development (Chan-Ling et al. 1995; Sandercoe et al. 1999). Astrocyte migration precedes the formation of patent vessels during normal development in both the rat and cat retinas (Ling and Stone 1988; Ling et al. 1989). A close relation between astrocytes and the forming vasculature has also been demonstrated at the ultrastructural level, with astrocytic processes being shown to associate with both well differentiated and immature vessels during normal human retinal vasculogenesis (Penfold et al. 1990). Astrocytes play a role both in endothelial cell differentiation (Jiang et al. 1995) and in BRB function (Chan-Ling and Stone 1992). They contribute to the developing vascular complex at an early stage, contacting the collagenous matrix surrounding capillaries at a time coincident with the opening of the capillary lumen. The proximity of astrocytes to the differentiating vascular endothelium at the time of lumen formation suggests that these cells, as well as Müller cells, are ideally located for induction of the BRB during development (Bellhorn 1980;

28.7 Pericytes and the BRB

Hughes et al. 2000; Chan-Ling et al. 2004 a). Conversely, the endothelium may also influence astrocyte differentiation. Vascular endothelial cells have been shown to induce the expression of glial fibrillary acidic protein (GFAP) in astrocyte precursor cells from the rat optic nerve in vitro, an effect that is likely mediated by the production of leukemia inhibitory factor (Mi et al. 2001). Vascular segments are produced in excess relative to the metabolic requirements of neurons and glia during CNS development. The vascular tree subsequently undergoes substantial remodeling, during which excess capillary segments are pruned and major vessels are selected (Hughes and Chan-Ling 2000). It is not known whether vessel regression is associated with a transient, focal loss of the BBB. Segments of vessels at various stages of formation and regression in the retina lack occludin immunoreactivity (Morcos et al. 2001) (Fig. 28.2 A–D). Discontinuous occludin expression is most evident in remodeling vascular segments in regions of the retina central to the leading edge of vessel formation, where vessel retraction occurs. A small proportion of vessels at the leading edge of vessel formation, where endothelial cell migration takes place in association with vessel formation, also exhibit discontinuous occludin expression. These observations suggest that loss of continuous occludin expression is associated with the migration of endothelial cells during developmental remodeling of the vascular plexus, but that most of the endothelial cell migration associated with vessel formation is completed prior to the expression of occludin. These changes in occludin expression can be interpreted in at least three ways. First, the loss of occludin expression reflects a focal, transient breakdown of the BRB. Second, such a loss occurs only in vessel segments that lack a lumen. Third, the absence of occludin in forming or regressing vascular segments is an artifact attributable to the inability of the monoclonal antibody used for these studies to detect all forms of occludin.


The capillary wall consists of an endothelial tube ensheathed by pericytes, which lie within the basal lamina of the vessel; and mural cells have been shown to be associated with newly formed retinal vessels (Chan-Ling et al. 2004 a, b). Postulated functions of pericytes include vessel stabilization, control of capillary blood flow or macrophage activity, and modulation of vascular permeability in the CNS (see Chapter 5). In the CNS, pericytes express BBB-specific enzymes, such as a specific isoform of aminopeptidase N (pAPN; Kunz et al. 1994) and glutamyltranspeptidase (Frey et al. 1991). In vitro studies showed that culture medium conditioned by a brain pericyte cell line increased the level of occludin expression in brain capillary endothelial cell lines (Hori et al. 2004). Furthermore, coculture of a brain endothelial cell line with pericytes triggered upregulation of BBB function (Dohgu et al. 2005). Mice that lack either the B chain of platelet-

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Fig. 28.2 (A–D) Light microscopic analysis of occludin expression in vessels of the rat retina during development. (A) and (C) show occludin expression, while (B) and (D) show the GS lectin appearance of the vascular segment in the respective fields of view. (A) and (B) show vascular segments at the leading edge of vessel formation. The majority of the vascular endothelial cells showed continuous occludin expression between adjacent vascular endothelial cells. The arrowheads demarcate the segment of the blood vessel, which lacks occludin

expression. The occludin-free segment in (A) appears to lack a vessel lumen and also shows a weak expression for GS lectin (B). The avascular retina is on the right of the field of view. The occludin-free segment shown in (C) has the characteristic appearance of a basal lamina when visualized with GS lectin (D). (E) H&E-stained transverse section of the region of the optic nerve head in a monkey. The retina and optic nerve head is in a unique position where CNS microvasculature is adjacent to leaky peripheral vascular beds,


derived growth factor (PDGF) or the b chain of the PDGF receptor manifest a reduced number of pericytes as well as edema and evidence of the immaturity of tight and adherens junctions in CNS vessels (as revealed by the patterns of occludin and VE-cadherin immunostaining, respectively) and increased transcellular transport. Immunostaining for other tight junction proteins was unaffected in these mice, however, and there was no morphological evidence of altered tight junction permeability. The junctional phenotype of these knockout mice was thus suggested to be a result of the increased expression of the vascular endothelial growth factor-A (VEGF-A), which was also apparent in these animals, rather than of the reduced number of pericytes (Hellstrom et al. 2001). The extent of pericyte coverage of retinal vessels is markedly greater than that of choriocapillaries (Tilton et al. 1985) or cerebral capillaries (Frank et al. 1987, 1990). The increased ratio of pericytes to endothelial cells in the retina has been proposed to account for the observation that the BRB is less vulnerable to acute hypertension than is the BBB (Laties et al. 1979). It has also been suggested that retinal vessels require an increased level of pericyte coverage in order to maintain a robust BRB, given that they exhibit a higher density of inter-endothelial cell junctions than do cerebral vessels (Stewart and Tuor 1994). The location of pericytes on the abluminal surface of the endothelium has also led others to postulate that pericytes may play a role in the vascular response to BRB breakdown (Sakagami et al. 1999). Given that these cells express tissue factor, they may also contribute to blood coagulation (Bouchard et al. 1997; Rucker et al. 2000).

Fig. 28.2 (continued) including the choroid. This continuity between structures with CNS and peripheral characteristics in the eye results in regions with an inherent breakdown of the bloodretinal barrier and immune activation. The region of the optic nerve head is one such region, making it prone to inflammatory attack in autoimmune diseases, such as multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). Optic neuritis is the clinical manifestation of the inherent weakness of the BRB in the region of the optic nerve head. (F) The peripapillary region of the control CBA mice is characterised by a marked increase in permeability due to the discontinuity of the glia limitations. The increased permeability is evidenced by the higher fluo-

rescence from the bisbenzimide around the optic disc. (G) HRP leakage in the optic nerve of control rats. Small arrows indicate meningeal surface. Retina (Ergun et al. 1997) shows HRP leakage from meninges in the lamina cribrosa (LC) region (large arrow) of control rats. (H) MHC class II+ cells with extensive processes (large arrow) in the meninges of control rats. The expression of MHC class II on the surface of a cell is indicative of its potential for antigen presentation. The constitutive expression of MHC class II in the meninges of the retrobulbar portion of the optic nerve, observed in control rats, likely contributes to the development of inflammatory lesions in this region in rats with EAE.

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28.8 Membrane Proteins of Tight Junctions

Tight junctions are important in the paracellular transport pathway and maintain cellular polarity (Brightman and Tao-Cheng 1993; Vorbrodt 1993). Occludin was the first integral membrane protein shown to be localized exclusively to tight junctions in both epithelial and endothelial cells (Furuse et al. 1993; Hirase et al. 1997). Occludin contributes to both the structure (Fujimoto 1995; Furuse et al. 1996) and function (Balda et al. 1996; McCarthy et al. 1996; Chen et al. 1997; Wong and Gumbiner 1997) of tight junctions and is expressed in a wide variety of ocular tissues, including the rabbit ciliary epithelium and iris vascular endothelium (Wu et al. 2000). Two additional integral membrane proteins, claudin-1 and claudin-2, were subsequently shown to be associated with tight junctions, with the former protein being more widely distributed than the latter (Furuse et al. 1998). Claudin-1 and claudin-2 have been shown to belong to a large family of proteins, with more than 20 members identified to date (Furuse et al. 1998; Morita et al. 1999 a, b; Simon et al. 1999; Tsukita and Furuse 1999; Mitic et al. 2000). Overall, the claudins are widely distributed in the body, but only claudin-1 and claudin-5 have been shown to be expressed by endothelial cells (Morita et al. 1999 a, b) Evidence from cultured cells suggests that claudin-1 and claudin-2 are primarily responsible for the formation of tight junction strands and that occludin is an accessory protein that plays only a minor role in strand formation (Furuse et al. 1998). Whereas ZO-1 is expressed by vascular endothelial cells of the CNS, its expression is poorly correlated with tight junctional complexity or with permeability of endothelial cell monolayers (see Chapters 1 and 4 for further information). 28.9 Localization of Occludin and Claudin-1 to Tight Junctions of Retinal Vascular Endothelial Cells

Information on the expression of occludin and claudins in the retinal vasculature is limited. Increased occludin expression appears to be associated with the development of CNS barrier properties. The extent of occludin expression in brain endothelial cells is thus markedly greater than that in endothelial cells of nonneural origin (Hirase et al. 1997); and downregulation of occludin expression correlates both with breakdown of the BRB in experimentally induced diabetes (Antonetti et al. 1998) and with neutrophil-induced BBB breakdown (Bolton et al. 1998). In human gliomas, expression of claudin-1, but not that of claudin-5, is downregulated in association with an increase in microvascular permeability (Liebner et al. 2000). In contrast, expression of claudin-5 and occludin was found to be downregulated in hyperplastic vessels (Liebner et al. 2000). The application of occludin and claudin-1 immunohistochemistry and immunoelectron microscopy to vessels of the rabbit eye has provided information on the molecular structure of tight junctions during vessel formation and retraction

28.10 Expression of Occludin by RPE Cells and Lack of Occludin Expression by Choroidal Vessels

of vascular segments as well as on the influence of various glial cells on junctional protein expression in different vascular beds (Morcos et al. 2001). Barrier properties and glial ensheathment of vessels were examined by intravascular injection of HRP and by immunohistochemistry, respectively. The distribution of occludin was also examined both in the vessels of the choroid, which lack barrier properties (Flage 1977; Morcos et al. 1999), and in the RPE. Both occludin (Fig. 28.1 B) and claudin-1 were detected at the junctions of adjacent vascular endothelial cells in the myelinated region of the rabbit retina. Both proteins were specific to vascular endothelial cells, not being expressed by any other cell type within the myelinated streak. In the adult rat retina, consistent with the known barrier properties of intraretinal blood vessels, occludin is expressed by endothelial cells of both the superficial and deep retinal plexuses (Chan-Ling et al. 2005). Consistent with the association of occludin expression with vessels that exhibit functional tight junctions, this protein was found to be expressed at a reduced level in vessels of the aged rat retina, which were also found to exhibit breakdown of the BRB, as revealed by intravascular perfusion with HRP (Chan-Ling et al. 2005). Immunoelectron microscopy showed that claudin-1 immunoreactivity occupied a slightly greater percentage of the area of tight junctions than did occludin immunoreactivity and that it also extended slightly into the cytoplasm of endothelial cells in the myelinated streak of the rabbit retina (Morcos et al. 2001). These observations suggested that the structural role of claudin-1 may differ from that of occludin. The sites of occludin and claudin-1 immunoreactivity were separated by similar regular intervals (79 nm and 82 nm, respectively), suggesting that the two proteins are localized to the same tight junction strands. These observations also suggested that the paracellular pathway, by which material is transported across tight junctions (Madara 1998; Tsukita and Furuse 2000), consists of the occludin- and claudin-1-positive tight junction strands and the regular intervals between these strands.

28.10 Expression of Occludin by RPE Cells and Lack of Occludin Expression by Choroidal Vessels

RPE cells of the rabbit were found to express occludin around their entire circumference, consistent with the function of these cells as a barrier separating the retina from the leaky vessels of the choroid (Morcos et al. 2001) (Fig. 28.1 C). The rabbit RPE also showed continuous claudin-1 expression, consistent with the formation of tight junctions between adjacent RPE cells (Fig. 28.1 D). Expression of these two tight junction proteins thus likely contributes to the functional boundary between the retina and the leaky blood vessels of the choroid. In contrast, occludin immunoreactivity was detected at only a low level and showed an irregular distribution along the vessels of the choroid (Fig. 28.1 E), a vascular bed that lacks BBB properties.

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28.11 Inherent Weakness of the BRB and Existence of Resident MHC Class II+ Cells Predisposes the Optic Nerve Head to Inflammatory Attack

Blood vessels in the retrobulbar optic nerve are permeable to intravascular tracers, such as Evans blue and HRP (Tso et al. 1975; Chan-Ling et al. 1992 a, b). This inherent weakness of the BBB has been suggested to be responsible for the susceptibility of this region to lesion formation in both experimental allergic encephalomyelitis (EAE) and multiple sclerosis (MS; Guy and Rao 1984; Hu et al. 1998 a, b). Indeed, regions of the CNS, such as spinal nerve root sheaths (Pettersson 1993), that lack an inherent BBB often show early accumulation of inflammatory cells in animals with EAE (Shin et al. 1995; Hu et al. 1998 a, b). In addition, the periventricular white matter is susceptible to the formation of plaques in individuals with MS (Adams 1977). These observations are consistent with the hypothesis that sites of inherent breakdown of the BBB are susceptible to lesion formation in EAE and MS. HRP and bizbenzimide leakage from the meninges are apparent only in the region of the lamina cribrosa in control animals (Fig. 28.2 F, G). However, intravascular injection of Monastral blue revealed no evidence of hemorrhage in the lamina cribrosa region of control animals. These observations show that this region has a mild weakness of the BRB, as indicated by leakage of HRP and bizbenzimide, but is not of a nature that results in franked hemorrhaging as would be evident by leakage of Monastral blue. This site of inherent weakness of the BRB is also characterized by the presence of a resident population of MHC class II+ cells. This resident population of MHC class II+ cells is present in the meninges surrounding the optic nerve and the lamina cribrosa region of the optic nerve in naive rats (Fig. 28.2 H). These cells possess extensive processes and are possibly the equivalent of the MHC class II+ dendritic cells previously detected in the choroid plexus of the eye (Forrester et al. 1994). The expression of MHC class II on the surface of a cell is indicative of its potential for antigen presentation (Lassmann et al. 1994). Expression of MHC class II is associated with the formation of inflammatory lesions in EAE (Molleston et al. 1993) and autoimmune diseases of the peripheral nervous system (Pollard et al. 1986). The choroid plexus of the eye is not a part of the CNS, nor does its vasculature show embryological or functional characteristics of CNS microvessels (Chan-Ling and Stone 1993). However, because of its proximity to, and continuity with, the meninges of the optic nerve (Fig. 28.2 E), the characteristics of the choroid are important in the development of optic neuritis. Therefore, inherent breakdown of the BRB and constitutive expression of MHC class II in the meninges of the retrobulbar portion of the optic nerve, observed in naïve subjects, predisposes this region to the development of inflammatory lesions in this region in rats with EAE and patients with MS.

28.12 Clinical and Experimental Determination of the Blood-Retinal Barrier

28.11.1 Compromised BBB Where CNS Meets Peripheral Vascular Bed

Other regions of inherent weakness of the BBB include the choroid plexus within the ventricles (Brightman and Reese 1969). Vessels of the ventricle are highly permeable due to their function in the formation of cerebral spinal fluid; and we also have demonstrated the existence of a substantial population of constitutive MHC class II+ cells in this region in naïve control adult JC Lewis rats (Ping Hu and Tailoi Chan-Ling, unpublished data). This region of the CNS is also prone to inflammatory attack during acute EAE. A third region of special interest is the region of the retina adjacent to the ciliary margin where the retina is adjacent to the leaky vascular bed of the ciliary processes, a bed responsible for secretion of the aqueous humor.


The eyes have been colloquially identified as the ‘window’ to the brain for many decades. This is because of the transparency of the ocular media, making it possible to view the retina in a living organism. In clinical studies, the integrity of the BRB can be evaluated by fluorescein angiography and vitreous fluorophotometry. These methods are able to provide qualitative and quantitative information of the BRB and in particular the exact location of the breakdown of the BRB. Further, the arterial/venous pairs that characterise mammalian retinal vascular tree makes it possible to determine whether it is an arterial or venous vessel that is compromised or whether it is a capillary-sized vessel that is affected. While these techniques are useful in the clinic, far greater details of the functional alteration of the BRB can be gained utilizing invasive procedures on experimental animals. The majority of methods employed involve intravenous injection of intravascular tracers prior to the sacrifice of the experimental animals. The eye is then enucleated and the retina dissected and examined as a retinal wholemount preparation (Chan-Ling 1997). The retina is unique in that it is only around 200–250 lm in total thickness. As such, it can be examined as a retinal wholemount where the entire vascular tree can be examined with the relationship to neurons and glia intact. This approach has made possible the detection of a very mild and transient breakdown in the BRB induced by activated T cells of nonneural specificity which would not be possible if studying the brain (Hu et al. 2000) as well as the early detection of a breakdown in the BRB in an experimental model of cerebral malaria (Chan-Ling et al. 1992 a). Experimental determination of the BRB involves the injection of intravascular tracers prior to sacrifice of the animal. Tracers frequently utilised include horse radish peroxidase with a molecular mass of 44 000 kDa, which is similar to that of plasma albumin (Fig. 28.3 A), lanthanum hydroxide, and FITC-conjugated dextran of various known molecular weights.

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Fig. 28.3 (A) The superficial plexus of the kitten retina in a postnatal day 12 kitten retina visualized by intravenous injection of HRP prior to sacrifice. The tightness of the BRB in this region close to the optic disc is evidenced by inherent peroxidase activity of the red blood cells and HRP within the vessel lumens. The absence of HRP in the retinal parenchyma results in a tissue background that is almost transparent when the BRB is intact. This is in marked contrast to that seen in (B) and (C), where the BRB is breached.

(B) Lack of barrier properties in proliferative neovasculature in the kitten model of retinopathy of prematurity. The panel shows the extent of the neovascular rosette at 5 days after return of the kitten to room air, demonstrated via intravenous injection of HRP prior to sacrifice. The vessels at the edge of the rosette and throughout the retinal parenchyma are leaky. (C) At 10 days after return of the kitten to room air, the loss of HRP from within the vessel lumen is still evident, shown here in a mid-peripheral region of the retina.


The permeability of retinal vessels can be assessed, following perfusion with HRP, a long-established tracer used in studies of the BBB (for a review, see Broadwell 1989) either at the light or electron microscopic level. Briefly, HRP (200 mg kg–1 body mass in 1 ml of physiological saline) is injected into the tail vein 20 min prior to sacrifice. The retinas are dissected and then incubated in buffer containing diaminobenzidine, to visualize the reaction product. It can be mounted and examined with a transmitted light microscope at this stage or further processed for electron microscopy (Chan-Ling et al. 1992 b). Examination at the light microscopic level offers significant information of the exact sites of breakdown of the BRB, including the types of vessels affected as well as the region of the retina affected (Fig. 28.3 B, C), whereas studies at the ultrastructural level permits the localization of sites of HRP leakage with the morphology of the junctional complexes in the same vessel. The modified trichrome technique developed in our laboratory (Chan-Ling et al. 1992 a) has proven a useful tool for determination of the BRB. This technique involves the intravenous administration of Monastral blue, a colloidal dye, 2 h prior to sacrifice, followed by the intravenous injection of bisbenzimide and Evan’s blue 30 min prior to sacrifice. Only small quantities of dyes are injected, taking care not to increase the total blood volume, as small changes in blood volume can be fatal in fragile experimental animals. Monastral blue is a colloidal dye, the particles of which coat, or are ingested by, activated monocytes (Neill and Hunt 1992). It is

Fig. 28.3 (continued) HRP is seen both within the vessel lumen and as a brown reaction product in the parenchymal tissue. (D–E) The barrier properties of various retinal vascular beds demonstrated utilising the modified Trichrome technique. (D) The inner vascular plexus in control CBA mice visualized with intravascular perfusion of Evan’s blue and Bisbenzimide (double exposure). The nuclei of retinal vascular endothelial cells are visible as pale blue nuclei. The intravascularly injected Evan’s blue is bound to plasma albumin. All Evan’s blue is retained within the vessel lumens, indicative of the tight barrier properties of intra-retinal vessels in control CBA mice. (E) Where the BRB is intact, intravascular perfusion of bisbenzimide (a nuclear stain) results in staining of only vascular endothelial cells and intravascular leukocytes. Endothelial cells in arteries have a characteristic elongated morphology, whereas venous endothelial cells have a rounded morphology. Where the BRB is breached, resulting in

leakage of the bisbenzimide, retinal somas adjacent to the vessels are also lightly stained, as seen in (G). (F–H) The same field of view of a retinal vein, utilising the modified trichrome technique, at day 3 post-induction with ovalbumin (OVA)-activated T cells combined with intraocular injection of OVA. The intensity of the breakdown of the BRB, accumulation of monocytes, and franked haemorrhaging evident in these panels is typical of that observed in antigen-specific inflammatory responses. Arrows point to the same segment of the vein in each field of view and mark the location of adherent monocytes. (F) Evan’s blue show sites of leakage of plasma albumin. (G) Hoechst stain show endothelial nuclear morphology and leukocyte adhesion in the affected region of the retinal vein. (H) Monastral blue, Hoechst stain and transmitted light microscopy show the site of franked haemorrhaging as blue particulate matter on the outside of the vessel wall.

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also used to detect sites of marked breakdown of the BRB, as in frank hemorrhage. Briefly, rats are injected intravenously with 1 ml of 3% (w/v) solid Monastral blue solution in 0.9% (w/v) sodium chloride, 2 h prior to sacrifice, taking care to sonicate the dye immediately before injection, to disrupt aggregates. Evan’s blue binds strongly to serum albumin (68 500 kDa) (Wolman et al. 1981); and its distribution after intravascular injection reflects the distribution of serum albumin (Radius and Anderson 1980). Bisbenzimide, also known as Hoechst stain is a nonspecific DNA marker. Where the BRB is intact, intravascular injection of the dye restricts its access to contents within the vessel lumen, resulting in the nuclear staining of vascular endothelial cells as well as intravascular leukocytes. Where the BRB is breached, the bisbenzimide leaks out of the vessel lumen, thus exposing all retinal somas, resulting in the staining of neurons, glia, and microglia alike. The retinas are dissected (Chan-Ling 1997) and placed in fixative for 20 min prior to mounting and examination under a fluorescent microscope. Evan’s blue is observed under green fluorescence microscopy (excitation filter 545 nm, barrier filter 590 nm) and appears red (Fig. 28.3 D, F). The Hoechst stain is examined under ultraviolet fluorescence microscopy (excitation filter 360 nm, barrier filter 455 nm) and appears light blue (Fig. 28.3 D, E, G, H). Transmitted light microscopic examination is used to examine the rheologic aspects of the blood column; and, in particular, erythrocytes can be visualized easily through their brown hemoglobin and monocytes by their phagocytic granules of Monastral blue and their characteristic nuclear morphology (Fig. 28.3 H). The modified trichrome technique has been successfully applied for the study of the BRB in a number of experimental models (Chan-Ling et al. 1992 a; Hu et al. 1998 a, b, 2000). Compared with histological sections of the brain or retina, the retinal wholemount technique allows earlier detection and localisation of marginating monocytic cells, congestion, and small hemorrhages and is an extremely sensitive tool for the detection of even the mildest breakdown of the BRB. Other changes to the microvasculature, such as occlusion of vessel segments to plasma perfusion are only detectable using the modified trichrome technique in retinal wholemounts.

28.13 Conclusions

In humans and several other mammalian species, the BRB is composed of two parts because the retina has a dual vascular supply, the inner two-thirds of the retina being nourished by intraretinal vessels and the outer one-third being supplied by choroidal vessels. The barrier properties of the inner retina depend on the tight junctions between adjacent endothelial cells of the intraretinal capillaries, whereas those of the outer retina depend on the tight junctions between adjacent cells of the RPE. These two barriers have been described as the inner and outer BRB, respectively. The retina is an extension of the CNS and, as a result, the characteristics of the blood-retinal interface is similar to that of the blood-brain interface, includ-

References

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Subject Index a Ab amyloidose 279 A cell 151 ABC – superfamily 432 – transporter 425, 442 absorption, distribution, metabolism and excretion, see ADME Acanthamoeba 680 Acanthamoeba castellanii 683 acetate 573 acetylcholine 274, 613, 654 acidic fibroblast growth factor (aFGF) 18 acidosis 653 acoustic stimulation 574 actin 443 actin cytoskeleton 622 – mediator of cerebral edema 626 – paracellular transport 626 – pinocytotic vesicle formation 626 – regulated permeability 626 – tight junction 626 – transcellular flux 626 – zona occludens 626 active – influx/efflux 418 – transport 423 acute – lymphoblastic leukaemia (ALL) 445 – trauma 612 ADAM (a disintegrin and metalloproteinase) 317 ADAMT (a disintegrin and metalloproteinase with a thrombospondin motif) 317 addiction 580 adherens junction 15 adhesion molecule 117, 181, 290 ADME 404, 424, 436 – property 404, 421

adrenomedullin (AM) 53 advanced glycation end-product 90 aerobic glycolysis 578 African trypanosomiasis 672 ff., 689 ff. agent, HIV-associated 440 agrin 23, 96 AIDS 275 ff., 325, 693 f. AKT signal pathway 46 albumin 90, 654 – extravasation 242 ALK-1 212 alkaline phosphatase (ALKP) 211, 214, 241, 321 Alzheimer’s disease (AD) 277 ff., 440, 448, 480, 486, 612 ff., 659 – inflammatory response 613 American trypanosomiasis 689 ff. amino acid 654 f. – transporter 313 aminopeptidase A (APA) 321 aminopeptidase A (EC 3.4.11.7) 314 aminopeptidase M 314 aminopeptidase N 113 aminopeptidase N (EC 3.4.11.2) 314 aminopeptidase N pericytic pAPN 321 f. aminopeptidase N(M) (APN) 321 amoebae 678 amphiphilicity 436 b-amyloid (Ab) 448 b-amyloid aggregation 278 amyloid A precursor protein 278 amyloid peptide precursor (A4P) 613 b-amyloid protein 613 amyotrophic lateral sclerosis (ALS) 256 analgesia 485, 488 analgesic 487 androgen 481 Ang-1 26, 122 Ang-2 122


726

Subject Index angioblast 17 angiogenesis 17, 21, 28, 46, 49, 52, 61 ff., 120, 151, 321 f. – androgen-induced 63 – estrogen-induced 63 – persistent 62 angiogenesis-neurogenesis interaction 65 angiogenic factor 47 f. – neuroglia-derived 47 angiogenic vessel 42, 440 angiopoietin-1 (Ang-1) 19, 119, 211 f. angiopoietin-1 (Ang-1/Tie-2) 51 angiopoietin-2 (Ang-2) 19 Angiostrongylus cantonensis 681 angiotensin I 315 angiotensin II 315, 321 f. angiotensin III 321 angiotensin-converting enzyme (ACE, EC 3.4.15.1) 314 f. Anisakis 678 annexin-1 293 anorexia 274 ff. antibody 179 anticancer agent 435 antigen presentation 169, 172 antigen-presenting cell (APC) 138, 169 antioxidant 656 AP-1 299 A4 peptide 613 apolipoprotein E (apoE) 612 ff. apoptosis 216 Ab protein 614 – expression during ischemia 625 aquaporin (AQP) 621 – AQP1 216 – AQP4 94, 96, 98, 209 ff., 212 ff., 218, 224 f. – AQP9 218 – expression 216, 224 – expression during ischemia (AQP4) 625 – function 216 – water channel 197 arachnoid 140 ARD1 44 aromatic l-amino acid decarboxilase (AADC) 592 aromaticity 436 arteriole 112 f., 655 – vasoconstrictive response 655 artificial neural network (ANN) 419 f. ascariasis 678 ascorbate 653

astrocytoma 224 astrocyte 26, 78, 83, 90, 94, 110, 118, 120, 131, 209 f., 225 ff., 239, 320, 322, 357, 385, 431, 466, 486, 686, 691 ff. – activation 215 – aquaporine 624 – bipolar 189, 195 – boundary layers 195 – co-culture 225 – differentiation 49, 195 – endfeet 209 ff. – functional polarity 197 – inductive molecule 199 – morphology 193 – multipolar 195 – perivascular end feet 198 – plasticity 195 – polarity 194 – precursor cell 191 – protective of endothelium 624 – protective of neuron 624 – reporter molecule 573 – secondary barrier 624 – secreted molecule 200 – secretory function 199 – signaling molecule 199 – spacing 195 – territory 195 astrocyte-endothelial co-culture 225 astrocyte-endothelial signaling 202 astrocyte-to-neuron lactate shuttle 578 astrocytic – endfeet 23 – foot process 13 astrocytoma 216 astrocytosis 691 ATP 66 f., 566 ATPase 654 ATP-binding cassette (ABC) 405, 432 ATP-binding site 433 autoimmune 173, 255 – neuroinflammation 181 autoimmunity 179 AZT 475

b Babesia 678 bacteria 606 Balamuthia mandrillaris 683 barbiturate 487 barrier permeability 21 barriergenesis 28, 41 ff. basal lamina 117, 129

Subject Index baseline 565 – activity 578 basement membrane 110, 171, 655 basic fibroblast growth factor (bFGF) 18, 42, 144 BBB see blood-brain barrier BDNF 62 ff. behavior circuit 580 bEnd3 227 Benton’s visual retention test 659 benzylpenicillin 478 beta-adrenergic receptor 657 17beta-estradiol (E2) 298 bi-dimensional 384 bioavailability 403 blood-brain barrier (BBB, blood-brain interface) 11, 41, 49, 51 f., 77, 109, 143, 189, 209 ff., 247, 265 ff., 287, 313, 321, 357, 375, 403 ff., 431 ff., 444, 446, 463 f., 501 f., 552, 603 ff., 611, 614, 619, 621, 649 ff., 671 ff. – aquaporin 4 621 – astrocyte 621 – barrier function 654 – BBB+ 422 – BBB– 420, 422 – biochemical change 656 – biophysical change 656 – damage 215 – development 45, 209 – diabetes-related changes in transport 651 ff. – differentiation 212 – dysfunction 27, 612 – enzyme 466 – function 650 – functional change 649 ff. – functioning 209 – hemodynamic change 655 – induction 199, 201 – inflammation 351 – in silico prediction model 403 – insulin crossing 269 – integrity 658, 693 – in vitro blood-brain barrier 337 – in vivo study 337 – maintenance 27, 210 f., 216 – maturation 21 – pathophysiology 272 – permeability 269, 318, 431, 441 f., 612, 654, 684, 693 – permeation 403, 414, 418, 422 – phenotype 201

– property 211 ff., 224 f., 389 – transport 272, 274 f. – transport function 654 – transport system 265 – water transport 211 blood-brain barrier model 384, 421 – astrocyte conditioned medium 343 – brain vessel 338 – capillary 338 – coculture 347 – confluent monolayer 347 – species 346 – transport 349 blood-brain interface see blood-brain barrier blood-brain transfer 523 ff., 535 blood-cerebrospinal fluid 552 – barrier (BCSFB) 265, 432 blood flow 115, 536 ff., 540 ff. blood-liquor barrier 324 blood-oxygen level-dependent (BOLD) 557 blood-testis barrier 438 blood-tissue barrier 438 Bluthirnschranke 175 bone marrow transplantation 168, 174 bradykinin 315, 323 brain 49, 61 ff., 431, 438, 686 – activation 565 – active influx 405 – adult 61 ff. – angiogenesis 25, 41 ff., 212 – capillary 465 – capillary endothelial cell (BCEC) 431 – development 42 ff. – disease 325, 440 f. – drug 448 – endothelium 199 – endothelium function 202 – endothelium receptor 202 – glucose utilization 274 – gonadal steroid 62 – imaging methodology 554 – injury 612 – insulin-resistant state 279 – ischemic 215 – malignant tumor 440 – maturation 274 – metastasis 441 – microenvironment 49 – microvasculature 313 ff., 321 – nutrient transport 210 – oedema 612 – oxygen 523 – oxygenation 49

727

728

Subject Index – pathology 445 – perivasculature 130 – resolution 625 – response 49 – substrate 448 – targeted redox analog (BTRA) 489 – targeting 463 – tissue 529, 542 – trauma 255 – tumor 325 ff., 440 – vascular permeability 687 – work 565 brain edema 216 ff., 220, 619 f. – AQP1 625 – AQP4 624 f. – cytotoxic 620, 622 – during stroke 620 – ischemia 623 – Na-K-ATPase 622 – vasogenic 620 brain parenchyma cell 338 – astrocyte 347 – glial cell population influence 338 – pericyte 346 – regulation of blood-brain barrier properties 347 brain-targeting 473 brain vessel, maturation 212 brain water homeostasis 223 Brugia 678 bryostatin 1 614

c 45 Ca2+ 584 cadherin 88 – vascular endothelial 213 calcification 114 calcitonin gene-related peptide (CGRP) 53, 612 calnexin 443 canalicular multispecific organic anion transporter (cMOAT/MRP2) 442 cancer 576 capacitance 367 capillary 112 f., 542, 650 ff. – adventitial layer 130 – cerebral 650 ff – cortical 650 – lumen protein 210 – model 525 – profile 464 capillary-like structure 228 CAR 88

carboxypeptidase N 314 f. carrier 467 caspase-1 inhibitor 611 CATALYST pharmacophore model 424 caveolae 89, 442, 449 caveolar – protein PV-1 215 – trafficking 449 caveolin 251, 442 f. caveolin-1 90, 449 CCL 177 f. CCR 177 f. CD36 684 cell – culture 379 – differentiation 604 – immortalized 380 – line 380 – substrate contact 364 – surface antigen CD44 149 cell polarity, origin 196 cell-cell contact 357, 364 cellular – adhesion molecule 288 – basis of glucose utilization 572 – differentiation 388 – process 589 central nervous system (CNS) 49, 62, 265 ff., 278, 313, 325, 404, 419 f., 431, 438, 446, 448, 603 ff., 649 ff., 658, 672 ff., 682 ff., 689 ff., 692 f. – acid-base balance 657 – active drug 419 – BBB+ 419 f. – CNS+ 419 f. – CNS– 419 f. – disease 658, 215 – homeostasis 49, 210 – nonactive drug 419 – physiology 211 ceramide 449 cerebral – blood flow (CBF) 535, 566, 653 – cortex 239 – edema formation 622 – endothelial cell 357 – glucose metabolism rate (CMRglc) 653 – ischemia 326 – metabolic rate for glucose (CMRglc) 566, 568 – metabolic rate for oxygen (CMRO2) 566, 568 – metabolism 652

Subject Index – microvasculature 650 – microvessel 649 ff. cerebro-spinal fluid (CSF) 77, 211, 266 ff., 324, 603 ff., 613, 655 – diagnostic feature 603 – glucose 605 – protein concentration 605 cerebrovascular accident 659 ceruloplasmin 90 Chaga’s disease 689 ff. channel 209 characteristics for in vitro BBB model 347 – claudin 349 – JAM-A 349 – occluding 349 – TEER 349 – tight junction and parcellular permeability 349 – transcellular transport 350 – ZO-1 349 chemical delivery system 463, 468, 472 chemoattractant protein-1 (MCP-1) 252 chemokine 176, 181, 250, 608, 685 ff., 689 chemotaxis 607 chemotherapeutic agent 432 chemotherapy 440 cholesterol 443, 487, 656 choline 654 chondroitin sulfate 684 choroid plexus (CP) 12, 77, 129, 136, 194, 657 – epithelium 216 cilliary neurotrophic factor 249 cingulin 84, 89 circadian 254 circumventricular organ (CVO) 12, 77, 130, 196 clathrin 251 claudin 14, 84 f. claudin-1 26, 86, 242 claudin-5 319 CMRO2/CMRglc 568 – mismatch 578 CNS see central nervous system coculture 359, 385 cognitive – activity 576 – impairment 659 coimmunoprecipitation 443 collagen 140, 369 compartment model 533 f., 541 – insufficient oxygen delivery 541 – mitochondrial oxygen 533 f.

– oxygen transfer 528 – tissue oxygen 533 f. complement factor 179 computational model 410 computer tomography 556 connexin (Cx)43 141 contractility 115 cortical – freezing injury 574 – spreading depression (CSD) 328 Crane, R. K. 569 CSF see cerebro-spinal fluid CXCL 178 CXCR 177 f. CXCR4 216 cyclodextrin 484 cyclosporin A (CsA) 436 cysticercosis 672 ff. cytochrome 422 – oxidation 529 – oxydase 531 ff., 542 – P450 enzyme complex (CYP) 404 cytochrome c 651 cytokine 181, 247, 323, 436, 607 f., 683, 689 ff. – angiogenic 70, 212 – anti-inflammatory 611, 689 – molecule 290 – neurotrophic 62, 70 – proinflammatory 275 f., 318, 608 ff., 686, 689 cytokine-induced neutrophil chemoattractant-1 (CINC1) 250 cytoplasmic accessory protein 210 cytoskeleton 115

d DA transporter 582 DADLE 485 dehydroascorbate 653 ff. dementia 325, 475, 480, 580 dendritic cell 129, 138, 168, 173 2-deoxy-d-glucose (DG) 569 depression 659 descriptor for hydrophobic interaction 422 descriptor of polarity 421 destructive infection 694 dexamethasone 289 diabetes 274, 649 ff., – insulin-resistant 277 – noninsulin-dependent 279 – type 2 diabetes mellitus 277 diabetic neuropathy 660

729

730

Subject Index diapedesis 180 differentiation 115, 118 – of the BBB 25 diffusibility 541 diffusible tracer 568 diffusion – passive 431 – relation 534 digit span 659 diphtheria toxin receptor 501, 511 Dirofilaria immitis 678 dissociated GC 302 distributed model 531 – insufficient oxygen delivery 538 – mitochondrial 531 f. – tissue 531 f. DIV-BBB 384, 387 f. DOPA 592 DOPA-decarboxylase 93 dopamine 274, 582 dopaminergic system 580 DP71 223 Dracunculus medinensis 678 drug 433, 438, 445, 449, 654 – delivery system 470 – delivery to the brain 501 – of abuse 580 – resistance 671 – targeting 470 – transport 443 Duchenne muscular dystrophy (DMD) 220 ff. Duffy 176 dynamic – function 551 – movement 589 dyslipidemia 275 f. a-dystroglycan 96, 224 dystrophin 97, 198 – protein 220 f. dystrophin-associated protein (DAPs) 223 dystrophin-dystroglycan complex 96

e E3 ubiquitin ligase 44 EC see endothelial cell Echinococcus 678 ECIS 367 ECM 150 ECV304 292 edema 657 f., 685 efflux 650 f. – pump 432, 467

– transporter 441 f. EGF 323, 443 EGF/TGFa 66 Ehrlich, Paul 11, 175 electric cell-substrate impedance sensing (ECIS) 359 electrical resistance 13, 358 electrolyte transport 584 electrostatic interaction energy 417 encephalitis 325 endocytosis 253 – receptor-mediated 431 endogenous – fluorofluor 564 – substrate 436 – tumor necrosis factor (TNF-á) 683 endogenously derived ECM 368 endoglin 212 endothelial – secretion 448 – tight junction 385 endothelial barrier antigen (EBA) 656 endothelial cell (EC) 41, 77, 172, 179, 226 ff., 239, 250, 272, 287, 313, 321, 357, 431, 464, 609 ff., 650 ff. – activation 690 – capillary 440 – cerebral 318 – microviscosity 656 endothelial cell isolation 338 – aortic 338 – BBCEC 340 – brain capillary 338 – cerebral microvessel 338 – contamination with nonendothelial cells 344 – enzymatic 338 – filtration step 338 – glial cell 347 – immortalization 342 – MBCEC 340 – mechanical 338 – PECAM-1 346 – primary endothelial cell 341 – subculture 341 – von Willebrand factor 346 endothelial cell-selective adhesion molecule (ESAM) 15 endothelin A-like receptor 658 endothelin-1 (EN-1) 657, 688 endotoxin 275 energy consumption 652 energy currency 566

Subject Index enkephalin 484 eNOS 443 Entamoeba histolytica 678 ff. ependyma 192 f. – bipolar 193 ependymocyte 147 ependymoglia – derivative 196 – polarity 194 ependymoglial cell 191 f. – Müller cell 192 – tanycyte 192 Eph/ephrin family 48, 213 EphrinB-EphB families 19 epidermal growth factor 249 17-Epiestrol 299 epilepsy 440 EPO see erythropoietin erbB 66 erythropoietin (EPO) 42 ESAM 84, 88 Escherichia coli 606 E-selectin 690 estradiol 479 estrogen 287, 479 – receptor (ER) 297 ethylenediaminetetraacetic acid (EDTA) 654 exocytoplasmic face (the E-face) 13 experimental allergic encephalomyelitis (EAE) 177, 295 externally applied molecule 563 extracellular matrix 82,129, 313, 325, 357 – protein 119 extrinsic 563 ezrin 443

f factor inhibiting HIF-1 (FIH-1) 44 fatty acid 656 FDG-PET 576 feeding 255 FGF see fibroblast growth factor fibroblast 138 fibroblast growth factor (FGF) 47 – FGF/FGFR system 47 – growth factor receptor (FGFR) 239 – growth factor-2 (FGF-2) 211, 239 – growth factor-2 (FGF-2) 212 – growth factor-5 (FGF-5) 242 fibronectin 607 Fick principle 568 field-based method 421

filarial infection 678 filopodial extension 589 Fischer, Emil 569 flow-metabolism 535, 543 f. [18F]fluorodeoxyglucose (FDG) 569 [18F]fluoro-l-m-tyrosine (FMT) 592 flux capacity 540 [18F]FMISO 576 focal brain ischemia 242 fractalkine 176, 684 fractone 148 free energy of solvation 411 freeze-fracture technique 83, 211 FSH 481 functional – activity 565 – brain imaging 551 – magnetic resonance imaging 554 – MRI 557 funnel-shape 433

g GAG 176, 684 ganciclovir 476 f., 592 gap junction 112 gap junctional communication 141 GC therapy 296 GDNF 241 genetic – algorithm (GA) 414 – engineering 592 – function approximation (GFA) 413 geohelminth 678 GFAP see glial fibrillary acidic protein glia 385 glia limitans 172, 181, 191, 198, 209 – perivascularis 170 glial – activity 66 – cell 23, 313 – cell-derived neurotrophic factor 249 – endfeet protein 213 – limitans 138 – limiting membrane 218 – perivascular endfeet differentiation 213 glial fibrillary acidic protein (GFAP) 212 ff., 241, 438 f. glioblastoma 440 gliogenesis 61 glioma 220, 440 glioma C6 228 gliovascular unit 66 global method 567

731

732

Subject Index glucocorticoid hormone 287 glucose 209, 214 f., 277 f., 567 f., 605 – analog 569 – transport 652 ff. – transporter (GLUT-1) 16, 92, 313, 614, 660 – transporter insulin-sensitive 278 – transporter-1 protein 298 glucuronoxylomannan (GXM) 608 GLUT1 22, 241, 653 ff. glutamate – pool 573 – transporter 198 c-glutamyl transpeptidase (c-GT) 211, 241, 321 glutathione 93 Goldman, Edwin E. 12 Gombar-Polli rule 424 GR-1 684 granulocyte colony-stimulating factor (G-CSF) 301 granulomatous amoebic encephalitis 681 green tea 438 GRID 421 growth factor 46, 318, 323 Guillain Barré syndrome 605

h H+-ATPase 657 Haemophilus influenzae 606 HBEC 292 H-bond – accepting 436 – acceptor/donor feature 404 – property 411 HDL 90 heat-shock protein 288 hemodynamic change 655 hemodynamic force 634 – flow cessation 634 – myogenic tone 634 – pinocytotic vesicle formation 635 – reperfusion-induced hyperemia 634 – shear stress alteration 634 hemoglobin – saturability 541 – saturation 525 hemorrhage 685 ff. heparan sulfate proteoglycans (HSPG) 140 heparin-binding – epidermal growth factor-like growth factor (HB-EGF) 501

– growth factor 148 hexose transporter 653 ff. HIF see hypoxia-inducible factor HIF-responsive element (HRE) 44 highly charged polar surface area (HCPSA) 415 Hill – coefficient 529 ff. – equation 525 hippocampal formation 239 histamine 318 HIV/AIDS 693 HIV-1 180 HIV-associated dementia 440 homeostasis 118, 209 f., 220, 274, 278, 321, 686 hormone 654 – replacement therapy 479 horseradish peroxidase (HRP) 213, 651 housekeeping gene 566 HSP70 323, 443 HSP90 443 HSVEC 300 HT7-antigen 215 human – brain EC 381 – brain endothelial cell 292 – brain microvascular endothelial cell (HBMEC) 51 – brain tumor 28 – endothelial-like cell 292 – ether-a-go-go related gene (hERG) potassium channel 404, 422 – parasitic disease 671 ff. – saphenous vein endothelial cell 300 – umbilical vein endothelial cell 292 human-applicable carrier protein (CRM197) 511 HUVEC 292 hydrocortisone 241, 358 hydrogen ion concentration 584 hyperglycemia 274, 276 ff., 652 ff., 658 hyperglycemic stroke, brain edema 633 hyperinsulinemia 277 ff. hyperosmolarity 655, 658 hypoglycaemia 605, 652, 660 hypoinsulinemia 274 hypoxia 42, 46, 627 – ATP 627 – depletion during ischemia 627 – permeability 627 – production of autocoids (NO, O–2) during reperfusion 627

Subject Index – state of actin 627 hypoxia-inducible factor (HIF) 42 hypoxic – ischemia 69 – tissue 576

i ICAM-1 see intercellular cellular adhesion molecule IFN-beta 296 IFN-c 690 ff. IGF1 65 ff. Ik B 611 IL1-b see interleukin-1b IL-6 see interleukin-6 image pH 586 immobilized artificial membrane (IAM) 379 immune – clearance 684 – response 215, 611, 685 – system 681 immune-privileged 175 immunoconjugate 302 immunoglobulin G 654 immunoglobulin superfamily 685 immunosuppression 678 in silico predicition 403 in vitro blood-brain barrier model 28, 358, 378 increased glycation 649 infection 605 inflammation 93, 289, 609, 692 inflammatory – cell 685 – condition 605 – process 613 – response 603 ff. – state 275 f. inhibitory domain 44 innate immunity 170 insulin 90, 265 ff., 274 ff., 431, 653 – CNS 274, 279 – dysregulation 278 – pathophysiology 272 – receptor 443, 504, 508 – receptor desensitization 279 – resistance 275 ff. – resistance syndrome (IRS) 277 – sensivity 276 – signal transduction cascade 279 – TNF-induced resistance 276 – transport 272, 275 ff.

insulin-degrading enzyme (DIE) 278 f. insulin-like growth factor 1 249 insulin-resistant brain state 279 integral membrane protein 210 integrin 290, 685 – receptor 369, 607 b-integrin signaling 48 intercellular – adhesion molecule (ICAM) 290, 608 – cellular adhesion molecule 93, 613, 684 ff., 690, 694 ff. interferon 296 – gamma (IFN-c) 292, 318, 611, 684 ff. interleukin 248, 691 ff. interleukin-1 90, 607 ff. interleukin-1b (IL-1b) 292, 318, 323 interleukin-6 (IL-6) 211, 241, 276, 683 ff. interleukin-10 250 intermediate filament protein 113, 241 internalizing receptor 501 intracranial hemorrhage 605 intravital microscopy 651 intrinsic 563 – signal 587 inulin 654 invasive brain drug delivery strategy 504 ischemia 584 islet amyliod polypeptide (IAPP) 278

j JACOP 84, 89 JAM 84, 87 f. – protein 210 junctional adhesion molecule (JAM) 15, 291, 608 juxtavascular microglia 169, 172 f.

k kainic acid 584 Karnowsky, Morris 12 K+ channel 224 K+ concentration 209 ketoacidosis 658 ketone body 653 ketosis 653, 658 Kety, Seymour 568 Kir 4.1 219 knockout mouse 448 Kolmer cell 130 Kolmer macrophage 136 Krogh cylinder 523 Krogh’s diffusion coeffizient 531 f. K+ siphoning 211

733

734

Subject Index K+ spatial buffering 220 kyotorphin 484, 487 f.

l lamina affixa 138 laminin 140, 369 LDL see low-density lipoprotein leakiness 27 Leishmania 681 leptin 660 leptomeninges 170 leu-enkephalin 485 leukemia inhibitory factor (LIF) 26, 49, 249 leukocyte 288, 685 ff. – migration 607 leukocyte-endothelial cell interaction 293 Lewandowsky, M. 12 LH 480 f. LHRH 480 lipid – peroxidation 656 – solubility 431 – transport 449 lipoaffinity 410 lipophilicity 404, 468, 485 lipopolysaccharide (LPS) 213, 224, 275, 291, 607 Listeria monoxytogenes 606 logP 468 lordosis 481 f. low-density lipoprotein (LDL) 90 – receptor-related protein (LRP) 320, 326 low-density lipoprotein (LDL) receptor 211 – family 320 – related protein 1 (LRP1) 501 Lowry, Oliver 568 LPS see lipopolysaccharide LRP1 receptor 509 LRP2 501 LRP2 receptor 509 luciferase 592 luminal membrane 438 luteinizing hormone 480 lysosome 116

m MAC-1 169 a-macroglobulin 315 macrophage 116, 129, 589, 607 ff., 694 – inflammatory protein 608 macrovascular disease 649 magnesium ATPase 657

magnetic resonance imaging 554, 556 MAGUK see membrane associated guanylate kinase major histocompatibility complex (MHC) 138 – class II 173 malaria 671, 689, 683 MAO see monoamine oxidase MAP kinase 658 – pathway 47 marker enzyme 359 marrow transplant 173 matrix metalloproteinase (MMP) 314 ff., 358 maturation 122, 580 mature BBB 25 MCAO 480 MCP-1 see chemotactic protein-1 or monocyte-chemoattractant protein-1 MDR 433 MDR1 gene 432 MDR1/ABCB1 444 – single nucleotide polymorphismus (SNP) 444 f. mdr1a gene 438 MDR-associated protein 442 mdx mouse 212 mdx3cv 220 mdxbgeo 220 MECA 32 antigen 215 mediator of EC permeability 631 – arachidonic acid 632 – bradykinin 632 – during ischemia 631 – during reperfusion 631 – endothelin-1 632 – histamine 632 – property 631 – vascular endothelial growth factor 632 membrane associated guanylate kinase (MAGUK) 15, 88 memory 278, 487, 659 meninges 129 meningitis 325, 604 ff., 681, 689 – acute bacterial 605 – aseptic (viral) 611 – integrity 611 meningoencephalitis 693 menopause 479 metabolic – brain image 567 – map 567 – X syndrome 277

Subject Index metabolism 523 ff. – cerebral 652 – oxygen 523 ff. metalloproteinase 145, 313 ff., 322 ff. – function 314 – types 314 metastasis 441 methylphenidate (MP) 582 methylprednisolone 291 MHC see major histocompatibility complex Michaelis-Menten equation 529 ff. microglia 78, 117, 181, 320, 322, 589, 611, 686, 691 ff. – activation 168, 172, 687 – resting 168 microglial cell 129, 167, 685 microinvironment 147 microneme protein 2 (MIC2) 694 microvascular – cell 381 – endothelial cell 239 – permeability 612 – wall 357 microvasculature 649 f. – brain 313 ff. – cerebral 649 ff. microvessel 525, 528 ff., 531, 542, 614 – cerebral 649 ff. – permeability 651 microviscosity 656 middle cerebral artery occlusion 242 migraine 328 mitochondria 529 ff., 614, 688 mitochondrial oxygen tension 525, 531 ff., 542 mitochondrion 541 MMP-9 145 Mn2+ 559 moesin 443 molar refractivity (MR) 414 molecular – imaging 551 f. – packaging 484 molecular weight (MW) 436 monoamine oxidase (MAO) 581 – MAO A 583 – MAO B 583 monocarboxylic acid 654 – transporter (MCT) 567 monoculture 382 monocyte 142, 167 – chemotactic protein-1 (MCP-1) 177, 608, 613

monocyte-chemoattractant protein-1 (MCP-1) 177, 294, 613 mono-dimensional 384 Monte Carlo simulation 412 mouse – brain capillary endothelial cell 361 – model 689 – model malaria 683 ff. – N-terminal acetyltransferase 1 (mNAT-1) 44 movement disorder 590 MPTP 592 MS 177, 180 multidrug – resistance transporter P-glycoprotein 431 ff. – resistance-associated protein Mrp1 93 – transport pump 405 multimodal assay 574 multiple linear regression (MLR) analysis 413 multiple sclerosis (MS) 176, 294, 325 ff., 605 myelination 688 myocardial infarction 660 myoinositol 649

n Na,K-ATPase 197, 211, 654, 657 NADPH 587 Naegleria fowleri 683 NAHD 474, 587 naloxone 486 natural killer (NK) 694 N-cadherin 26, 79 NDIV-BBB 390 near-infrared spectroscopy (NIRS) 562 negative BOLD response 557 Neisseria meningitidis 606 nematode 678 nephropathy 649 netrin-1 48 neural – progenitor cell 148 – stem cell 146 – tube, formation of 189 f. neuroactive substance 448 neurobiochemical 660 neuroblast 136 neuroblastoma cell (N2A) 230 neuroepithelial cell 190 neurofibrillary tangle formation 278 neurogenesis 61 ff., 147, 190 f., 278 – mitotic 67

735

736

Subject Index – persistent 65 neurogenesis (birth of neurons) 191 neurogenic niche 147 neuroglia 44, 46 neuroglial progenitor cell 41 neuroimmune function 210 neuroinflammatory response 612 neuron 320, 686 neuronal – differentiation 67 – GT1 228 – marker NFH (neurofilament H) 219 – percursor cell 239 – protection 210 neuronal polarization 196 neuron-specific enolase (NSE) 658 neuropathy 649 neuropeptide 274, 468, 484 neurophilin (NRP) 46, 48 neuroprotection 480, 483 neurotransmitter 220, 315, 613, 651 ff. – system 580 neurotrophin 216 neurovascular unit 94 neurulation 189 f. neutral aminoacid (NAAs) 214 neutral endopeptidase 24.11 (enkephalinase, NEP, EC 3.4.24.11) 314 NF-kB 299, 611 – pathway 611 NG2 chondroitin sulfate proteoglycan 68 nitric oxide (NO) 67, 657, 691 ff. nitric oxide synthase (NOS) 90, 92, 298, 487, 692 – synthase inhibition 67 nodule 114 noggin 67 nonCNS drug 419 nongenomic 289 non-metabolizable analog 572 non-receptor tyrosine kinase Lyn 215 norepinephrine 274 normoxic condition 44 NOS see nitric oxide synthase Notch receptor 48 nucleotide triphosphate diphosphohydrolase (NTDPase) 66 f. nucleotide-binding fold (NBF) 432 nutrient 650 ff.

o OAP see orthogonal arrays of intramembranous particles obesity 274 ff. obligatory fuel 567 occludin 14, 84 ff., 242, 295, 319, 650 f. oligodendrocyte 320 – precursor cell 191 3-O-methyl-d-glucose 572 Onchocerca volvulus 681 onchocerciasis 681 opiate 485 optical imaging 562 optico-acoustic imaging 564 organic anion transport protein (OATP) 423 orthogonal arrays of intramembranous particles (OAPs) 94, 96 ff., 211 ff. osmosensitive organ 219 oxidative – load 649 – stress 278 oxidized flavoprotein 587 oxygen 523 ff., 531 ff. – arterial 535 – arterial delivery 542 – capillary 539 f. – compartment model 528 ff., 541 – consumption 529 ff., 539, 542, 545 – diffusibility 532, 527 f., 544 ff. – distributed model 531 ff. – flow-metabolism 535 – flux 540 ff. – ischemic limit 546 – metabolism 523 ff., 535, 539 – mitochondrial 531 ff., 546 – regulatory gene 50 – saturation 537 – supply 538 – tension 41 ff., 49, 525, 528 ff., 539, 542, 684 – transfer 525, 528 oxygenation response 49 oxygen-dependent degradation (ODD) domain 44

p P100 659 p300 44, 659 p42/p44 mitogen-activated protein kinase (MAPK) 323 pain 660 – tolerance 659

Subject Index panendothelial cell antigen MECA-32 22 pAPN 113 f., 121, 327 paracellular pathway 404 paramagnetic agent 559 parasitic disease 671 ff. Parkinson’s disease 440, 445, 590 partition coefficient 468 f. passive diffusion 404, 410, 418, 431 pathophysiology 272 ff. PDGF see platelet-derived growth factor PDZ domain 84 PECAM-1 16, 88, 290 “peg and socket” contact 112 peptidase 485 b-peptide 613 pericyte 23, 26, 68 f., 78, 82, 90, 109, 129, 170 f., 209 f., 313, 321 f., 357 – marker 113 – microvascular 68 – positioning 110 – structure 110 perineural vascular plexus 17 peripheral neuropathy 660 perivascular – astrocyte 51 – astrocytic endfeet 209 – cell 321, 611 – macrophage 169 ff. – microglia 170 – pericyte 51 – plexus 41 f. – space 170 perlican 148 permeability 118, 180, 358, 389, 432 – capillary 318 – coefficient 469 – microvascular 215 – of the blood-brain barrier 651 peroxisome proliferator-activated receptorgamma (PPAR-gamma) agonist 280 PET 560 P-glycoprotein (P-gp) 16, 22, 79, 93, 211, 215, 313, 350, 404 f., 420, 422, 431 ff., 442 ff., 467, 469 f. – AHNAK 350 – endothelial adhesion molecule 351 – isoform 432 – localization 438 – MECA-32 350 – modulator 436 – polymorphismus 444 – structure 432

– structure-activity relationship (SAR) 434 ff. – subcellular localization 442 – substrate 435 – transport of LDL 347 pH response element 653 phagocytosis 117, 140, 170, 687 phagocytotic role 116 pharmacophore model 424 phenylarsine oxyd (PAO) 319 phospholipid 656 photon emission tomography 554 pial – macrophage 131 – transendothelial electrical resistance 624 – vessel BBB property 624 pig microvascular endothelial cell 358 pineal gland 239 pinocytosis 117, 140 – endothelial 215 pituitary gland 239 placenta 438 plasminogen activator (PA) 325 plasminogen/plasmin system enzyme 326 Plasmodium berghei 683 ff. Plasmodium falciparum 678 ff. Plasmodium malariae 678 ff. Plasmodium ovale 678 ff. Plasmodium vivax 678 ff. platelet-activating factor (PAF) receptor 606 platelet-derived growth factor (PDGF) 66 – growth factor B (PDGF-B) 119 f. – growth factor BB (PDGF-BB) 19 – growth factor-b (PDGF-b) 42, 52 pneumococcal surface protein (PspA) 606 pneumolysin 606 f. polar surface area 404, 410 polarizability 410 polymorphism 444 polymorphonuclear leukocyte (PMN) 604 ff. polyphenol 438 positive BOLD response 557 positron emission tomography 554 postcapillary venule 180 post-capillary venule 175 post-myocardial infarction 276 f. post-translational modification 656 potassium 574 – siphoning 211

737

738

Subject Index presenilin 279 primary culture 358 principal component analysis (PCA) 412 prodrug 468, 478 progenitor cell 61 ff. – microvascular influence 61 – mobilization 61, 66 f. – neural 66 – nitric oxide 67 – parenchymal 68 – post-ischemic mobilization 69 – purinergic signaling 66 proinflammatory mediator 607 protein glycation 649, 656 protein kinase C (PKC) 443, 614, 657 – hyperglycemia-induced PKC activation 634 – pathway 649 – permeability during hyperglycemia 633 – tight junction permeability 633 – vascular permeability 633 proteoglycan NG2 151 proton basicity 436 protoplasmic face (the P-face) 13 PSD95-disc large ZO-1 (PDZ) 223 pulsatile flow 388 purinergic signaling 66 pVHL-degradation pathway 44

q QSAR analysis, membrane interaction (MI-QSAR) 413 ff. QSAR model 403 ff., 424 quantitative – autoradiographic method 568 – autoradiography 560 quiescence 694

r [11C]raclopride 581 radial – glia (RG) 190 f. – glial cell 147 radioactive tracer 560 radionuclide imaging 560 radixin 443 Raichle, M. E. 578 Ras/Raf/MEK/MAPK pathway 66 rat brain endothelial cell (RBE4) 294 receptor-mediated – drug delivery 506 – transcytosis 504

redox analog 489 Reese, Thomas 12 regression model 410 remodeling 122 remyelination 243 reperfusion injury 628 – cerebral edema 628 – nitric oxide 628 – peroxynitrite 628 – reactive oxygen species 628 – vascular permeability 629 reporter molecule 563 resistance 367 resting state 565 retinal – Müller cell 97 – pigment epithelial cell 592 – pigment epithelium (RPE) 194, 197 retinopathy 649 reversal agent 436 RG2 228 Risau, Werner 17 rosiglitazone 279 rostral migratory stream (RMS) 191 rotable bond 404

s S100B 658 scaffolding domain 443 Schistosoma 678 Schistosoma haematobium 680 schistosomiasis 681 seizure 485, 487 – threshold 220 SEL 147 selectin 290, 684 sensory threshold 660 serine peptidases dipeptidyl peptidase II and IV (DPP) 314 f. serum – effect 362 – withdrawal 360 sexual – behavior 482 – dysfunction 481 shear stress 386 f. – laminar 386 – pulsatile 386 signaling pathway 212 single nucleotide polymorphismus 444 site-targeting index (STI) 470 f. SMAD 212 SMAD6 212

Subject Index small – interfering RNA 437 – ubiquitin-related modifier (SUMO) 44 f. smoker 583 smooth muscle – actin (SMA) 113 – cell 112, 115 – a-smooth muscle actin (a-SMA) 114 Sokoloff, Louis 568 Sols, A. 569 soluble E-selectin (s-ELAM-1) 613 solvent-accessible surface area (SASA) 412 sonic hedgehog (Shh) 48 sorbitol 649 sparganosis 678 spatial resolution 555 SPECT 560 sphingomyelin (SM) 450 sphingomyelinase 450 spinal cord injury 255 sprouting 121 src-suppressed C kinase substrate (SSeCKS) 26, 50, 52, 211 SSeCKS see src-suppressed C kinase substrate stabilization 122 stem cell 62 steroid 480 Streptococcus pneumoniae 605 f. streptozotocin (STZ)-induced diabetic 650 stroke 255, 479, 619, – hyperglycemic 633 stromal-derived factor-1 (SDF-1) 216 Strongyloides stercoralis 678 structure-activity relationship (SAR) 434 ff. subarachnoid hemorrhage 605 subependymal basal laminae 148 subgranular zone (SGZ) 65 substrate 435 SUBSTRUCT 420 subventricular zone (SVZ) 191 sucrose 651, 654 superparamagnetic iron oxide (SPIO) 559 support vector machine (SVM) 421 supraoptic nucleus 146 surface area 436 a-syn–/– mouse 220 a-syntrophin 223 a1-syntrophin 96 f.

t Taenia solium 678 ff. tanycyte 12, 194 targeting 463 targeting enhancement factor (TEF) 470 f. targetor (T) 472 tau phosphorylation 278 T-cell 215, 293, 318, 686 TEER see transendothelial electrical resistance temperature gradient 564 temporal discrimination 555 TFG see transforming growth factor T-helper – cell response 684 f. – lymphocyte 688 therapeutic index (TI) 472 thermal imaging 564 thrombospondin 684 thrombospondin-1 (TSP-1) 53 thymidine kinase 592 thyreotropin-releasing hormone (TRH) 484, 486 Tie-1 212 Tie-2 19, 122 tight junction (TJ) 11, 49, 77, 82 ff., 85 ff., 88, 92 f., 118, 172, 175, 179, 181, 196, 294, 318, 325, 357, 431, 609, 650 ff. – endothelial 201, 210, 213 – epithelial 201 – formation 211 – modulation 202 tissue inhibitor of metalloproteinase (TIMP) 315 ff. TIMP 322 ff. tissue-type plasminogen activator (t-PA) 326 T lymphocyte 143, 691 TNF see tumor necrosis factor Toll-like receptor 4 170 Tower 569 toxin 650 Toxocara canis 678 Toxoplasma gondii 694 toxoplasmosis 672 ff., 692 ff. – destructive infection 694 – quiescence 694 – transgression 694 trafficking 253 transactivation 288 – domain 44 transcellular transport 630

739

740

Subject Index – – – – –

actin depolymerization 631 during ischemia 630 during reperfusion 630 mediator of cerebral edema 631 pinocytosis in vascular permeability 630 transcription factor 611 – NFkB 323 transcytosis, receptor-mediated 405 trans-differentiation 129 transendothelial electrical resistance (TEER) 13, 318 f., 379, 387, 389, 465 transferrin 90, 431, 501 – receptor 215, 313, 504, 506 transforming growth factor (TGF) 48, 322 – alpha (TGFa) 251 – beta (TGF-b) 211 f., 611 – beta 1 (TGF-b1) 53, 119, 121, 144, 241 transgression 694 transport 248, 313, 572, – activity 438 – b-amyloid (Ab) 448 – carrier-mediated 405 – glucose 214 f. – multidrug transport pump 405 – system 376 transporter 423, 438, 442, 466 f., 553 – ATP-binding cassette (ABC) 432 – endothelial 214 f. – for low-density lipoprotein 504 – glucose 209, 660 – GLUT-1 653 ff. – hexose 653 – insulin-sensitive glucose 278 – ketone 653 – multidrug resistance 431 ff. transrepression 289 Transwell system 382, 407 trematode 678 TRH 486 Trichinella spiralis 682 tri-culture 120 [131I]trifluoroiodomethane 568 trigonelline 474 Trojan horse 174 Trypanosoma brucei gambiense 689 Trypanosoma cruzi 689 ff. trypanosome 692 trypanosomiasis 689 tumor necrosis factor (TNF) 276, 576 – receptor-2 687

– secretion 216 – TNF-a 292, 318, 323, 684 ff., 690 ff. – tumor necrosis factor alpha (TNFa) 144, 248, 274, 607 ff. tunica adventitia 135 two-dimensional gel electrophoresis 656 type 1 diabetes 652 type 2 diabetes 658

u ultrasound-mediated imaging 564 urokinase-type plasminogen activator (uPA) 326

v variable surface glycoprotein (VSG) 690 vascular – basement membrane 655 – damage 688 – endothelial (VE)-cadherin 213 – route 504 – stability 119 vascular endothelial growth factor (VEGF) 18, 42, 51 f., 61 ff., 85, 90, 92, 120,122, 144, 212, 242 – HIF1a-mediated production 65 – hypoxia-induced 46 – isoform 46 – receptor 46, 443 – receptor 1 (VEGFR1, flt-1) 18 – receptor 2 (VEGFR2, flk-1/KDR) 18 vascularization 42 vasculogenesis 28, 120 vasoactive agent 116 vasocinstrictive response 655 vasoconstrictor 116 vasogenic brain edema 220 vasorelaxant 116 VCAM-1 290, 690, 93 VE-cadherin 88 VEGF see vascular endothelial growth factor ventricular zone (VZ) 190 venule 112 f. vessel assembly 120 aV-integrin deficient mouse 27 Virchow-Robin space 133, 170, 172 viscosity 656 vitamin C 653, 661 vocal control nucleus HVC 63 ff. VolSurf descriptor 421 voltage-sensitive dye 587

Subject Index

w

y

Walker A motif 432 Walker B motif 432 water – channel 216 – transport 216 weakly rectifying potassium channel Kir4.1 97 white blood cell (WBC) 603 f. Wnt signaling 19 World Drug Index (WDI) 420

yolk sac 167

x xenobiotic 448 X-ray computed tomography

554

z zidovudine 475 ZO-1 15, 26, 84, 87 f., 242, 650 ZO-2 15, 84 ZO-3 15, 84 zonula – occludens 210 – occludens-1 (ZO-1) 212 f., 294, 319, 650 f. z-VAD-fmk (benzyloxycarbonyl-Val-Ala-Aspfluoromethyl ketone) 611

741

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