Role of the Extracellular Matrix in Myelination of Peripheral Nerve JEWEL L. PODRATZ, ESTHER RODRIGUEZ, AND ANTHONY J. WINDEBANK* Molecular Neuroscience Program, Mayo Graduate School and Medical School, Rochester, Minnesota KEY WORDS myelin; laminin; basal lamina; integrin receptor; Schwann cells ABSTRACT Assembly of the extracellular matrix (ECM) has been tightly linked to compact myelin formation in the peripheral nervous system. We recently demonstrated that myelination of dorsal root ganglion (DRG) axons by Schwann cells may occur in the absence of basal lamina. We have now determined whether laminin deposition occurs around myelinating SC, even though basal lamina has not been assembled. DRG/SC co-cultures were prepared from E15 rat embryos and incubated in fully defined medium (B27) with and without ascorbic acid for 21–24 days. Cultures were stained with a rabbit anti-laminin antibody and examined by laser confocal fluorescence microscopy. Myeli- nation occurred in both groups. In the presence of ascorbic acid, there was dense even laminin staining around myelinating SC. In the absence of ascorbic acid, laminin staining was also present but was irregular and less dense. DRG and SC were co- cultured without ascorbic acid in the presence or absence of a function blocking anti- 1 integrin receptor antibody. The antibody completely inhibited myelination. Finally, DRG/SC co-cultures were prepared both with and without ascorbic acid and incubated under control conditions or in the presence of continual, gentle motion. Movement in the absence of ECM significantly inhibited myelination. This demonstrates that laminin deposition on the surface of SC but not ECM assembly is required for formation of compact myelin. ECM is required to provide mechanical stability during the process of myelination. GLIA 35:35– 40, 2001. © 2001 Wiley-Liss, Inc. INTRODUCTION Myelination in the peripheral nervous system (PNS) involves interactions between Schwann cells (SC), ax- ons, and the extracellular environment. Assembly of lamellar myelin occurs when SC are in contact with axons. Only axons with a diameter of 1 m initiate myelin assembly. In the mature PNS, unmyelinated axons are ensheathed by SC. SC have four major phe- notypes: immature, mature unmyelinated (ensheath- ing small axons), mature myelinated, and mature non- myelinated (mature cells that are not in contact with axons) (Mirsky and Jessen, 1996; Griffin et al., 1993). Both mature myelinated and unmyelinated SC are completely ensheathed by basal lamina. Immature SC and mature nonmyelinated SC share many character- istics including absence of basal lamina (Giannini and Dyck, 1990). The extracellular matrix (ECM) is critical for myeli- nation. It has two major components: the basal lamina surrounding SC and endoneurial collagen. Both are formed during embryonic development and both con- tain large amounts of collagen. Basal lamina contains type IV and type V collagen (Shellswell, 1979), laminin, fibronectin, entactin, and heparan sulfate proteoglycan (Bunge, 1993). The longitudinally oriented collagen fibrils in the endoneurial space are composed of types I, III, IV, and V collagen (Shellswell, 1979; Carey, 1983). Numerous studies have demonstrated that ECM for- mation and myelination are tightly linked (Bunge, 1993). The major structural protein in both basal lam- ina and endoneurial fibrils is collagen. When collagen synthesis is inhibited, ECM cannot be assembled and myelination cannot proceed. Synthesis and assembly of *Correspondence to: Anthony J. Windebank, Mayo Clinic and Foundation, 1501 Guggenheim Building, 200 First Street SW, Rochester, MN 55905. E-mail: windebank.anthony@mayo.edu Received 20 February 2001; Accepted 30 April 2001 GLIA 35:35– 40 (2001) © 2001 Wiley-Liss, Inc.