A first-time collection of cutting-edge morphologic, permeability, transport, in vitro, and molecular techniques to study the workings of the blood-brain barrier in health and disease. Described in step-by-step detail, each readily reproducible method includes an explanation of the theoretical basis of the method, a discussion of its advantages and limitations, notes on avoiding pitfalls, and examples of results obtained using the technqiue. These powerful tools can be used to isolate undiscovered molecules, deepen our understanding of their role in health and disease, and identify targets for future gene therapy and therapeutic intervention.

Preface Contributors 1 Morphology and Molecular Properties of Cellular Components of Normal Cerebral Vessels 3 2 Studies of Cerebral Vessels by Transmission Electron Microscopy and Morphometry 37 3 Freeze-Fracture Studies of Cerebral Endothelial Tight Junctions 51 4 Studies of Cerebral Endothelium by Scanning and High-Voltage Electron Microscopy 67 5 Detection of Endothelial/Lymphocyte Interaction in Spinal Cord Microvasculature by Intravital Videomicroscopy 83 6 Pathophysiology of Blood-Brain Barrier Breakdown 97 7 Pial Vessel Permeability to Tracers Using Cranial Windows 121 8 Blood - Brain Barrier Permeability Using Tracers and Immunohistochemistry 133 9 Ultracytochemical Studies of the Compromised Blood - Brain Barrier 145 10 Immunogold Detection of Microvascular Proteins in the Compromised Blood - Brain Barrier 161 11 Measurement of Blood - Brain and Blood - Tumor Barrier Permeabilities with [[superscript 14]C]-Labeled Tracers 177 12 A Review of Blood - Brain Barrier Transport Techniques 193 13 In Situ Brain Perfusion Technique 209 14 Brain Efflux Index Method: Characterization of Efflux Transport Across the Blood - Brain Barrier 219 15 Transport Studies Using Membrane Vesicles 233 16 Drug Transport Studies Using Quantitative Microdialysis 249 17 Single-Pass Dual-Label Indicator Method: Blood-to-Brain Transport of Glucose and Short-Chain Monocarboxylic Acids 265 18 Protein Transport in Cerebral Endothelium: In Vitro Transcytosis of Transferrin 277 19 Transport Across the Choroid Plexuses In Vivo and In Vitro 291 20 An Overview of In Vitro Techniques for Blood - Brain Barrier Studies 307 21 Isolation and Characterization of Human Brain Endothelial Cells 325 22 Human Brain Microvessel Endothelial Cell and Leukocyte Interactions 337 23 Development and Characterization of Immortalized Cerebral Endothelial Cell Lines 349 24 Isolation and Characterization of Retinal Endothelial Cells 365 25 Isolation and Characterization of Cerebral Microvascular Pericytes 375 26 Molecular Biology of the Blood - Brain Barrier 385 27 Blood - Brain Barrier Genomics 401 28 Dynamic In Vitro Model of the Blood - Brain Barrier: Gene Profiling Using cDNA Microarray Analysis 419 29 Phage Display Technology for Identifying Specific Antigens on Brain Endothelial Cells 435 30 mRNA Detection in Cerebral Vessels by Nonradioactive In Situ Hybridization 451 31 Proteomics of Brain Endothelium: Separation of Proteins by Two-Dimensional Gel Electrophoresis and Identification by Mass Spectrometry 465 32 Immunoblot Detection of Brain Vascular Proteins 479 33 Immunohistochemical Detection of Endothelial Proteins 489 34 Cerebral Vascular Function in Genetically Altered Mice 505 35 Generation of Transgenic Mice by Pronuclear Injection 513 36 Mouse Genome Modification 529 Index 545