This volume, Neuromethods: Extra-cellular Matrix, provides comprehensive procedures for analyzing the extracellular matrix in native, injured, and engineered neural tissues. The target audience is wide-ranging, targeting all levels of scientific and bioengineering researchers in the broad fields of neurobiology, development, medicine, and bioengineering. Each chapter is written by leading experts and presents established protocols in a concise format, encompassing current technologies as well as methods developed over years of research. Each chapter provides an introduction to the method, a listing of the materials and equipment, step-by-step protocols, and a Notes section with troubleshooting tips, supplemental details and alternative approaches, as well as a list of references for further information.
This volume is divided into four parts, each focusing on different aspects of the extracellular matrix and the nervous system. The first part focuses on methods to analyze native tissue, including basal lamina in the brain and neuromuscular junctions as well as perineu-ronal nets of the central nervous system. The second part presents in vitro models for investigating cell-extracellular matrix interactions in a variety of contexts. For example, it presents extracellular matrix proteins and proteoglycans as uniform or gradient substrata, high-throughput methods to analyze neurite outgrowth, as well as three-dimensional cultures of tissue explants and isolated cells. The third part provides protocols to investigate the role of the extracellular matrix in nervous system injury, degeneration, and regeneration, including brain tumors and spinal cord injury. This part also includes methods to modify glycosyl-ation of extracellular matrix proteins in cultured neural stem cells. The final part of this volume centers on therapeutics and engineered systems, including analysis of extracellular matrix proteins in tissue engineering constructs, cultivation of astrocytes on aligned micro-fibers, microfluidic devices to study neural stem cell chemotaxis, and cutting-edge hydrogel biomaterials for cell culture and transplantation.
We would like to thank all contributing authors for their time spent preparing the chapters and for sharing their valuable expertise. We would also like to thank the series editor, Dr. Wolfgang Walz, for inviting us to edit this volume and for his constant enthusiasm for the work. We would also like to thank those at Springer, especially Patrick Marton, who have strongly supported this volume with helpful advice through the entire process. Baltimore, MD, USA Jennie B. Leach; Elizabeth M. Powell

Preface v

Series Preface vii

Contributors xi

Part I Native Tissue

1 Immunohistochemical Characterization of Brain Neural and Vascular Basement Membranes 3

2 The Use of Synaptic Basal Lamina and Its Components to Identify Sites of Recent Morphological Alterations at Mammalian Neuromuscular Junctions 13

3 Perineuronal Nets: A Special Structure in the Central Nervous System Extracellular Matrix 23

Part II In Vitro Models

4 Isolation of Perinatal Mouse Cerebral Cortical Astrocytes 35

5 Techniques for Examining the Effect of Substratum-Bound Proteoglycans on Neurite Outgrowth In Vitro 45

6 High-Throughput Quantitative Assay for Analyzing Neurite Outgrowth on a Uniform Substratum: The Cell-Substratum Assay 57

7 Extracellular Matrix Components as a Substrate for Outgrowing Motoneurons 67

8 Replicating the In Vivo Environment: Organotypic and Submerged Three-Dimensional Culture Methods 79

9 Culturing Neurons, Glia, and Progenitor Cells in Three-Dimensional Hydrogels 91

10 Investigating Cell-ECM Interactions and ECM Synthesis in Three-Dimensional Hydrogels 101

Part III Injury, Degeneration, and Regeneration

11 Characterization and Analysis of Extracellular Matrix in Malignant Brain Itamors and Their Cellular Derivatives 113

12 Chondroitinase Gene Therapy for Spinal Cord Injury 139

13 Modulation of Neural Stem Cell Expressed Extracellular Matrix (ECM) by Targeting Glycosyltransf erases 151

Part IV Therapeutics ajsid Engineered Systems

14 Evaluating the Spatial and Temporal Protein Production in Neural Tissue Engineering Constructs In Vitro 163

15 Cultivation and Imaging of Astrocytes on Protein-Coated Fluorescent Topographies Constructed from Aligned PLLA Electrospun Fibers 181

16 Engineered Microdevices to Study and Manipulate Neural Stem Cell Chemotaxis 197

17 Hydrolytically Degradable Polyethylene Glycol (PEG) Hydrogel: Synthesis, Gel Foimation, and Characterization 211

Index 227

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