Murine spinal cord explants: A model for evaluating axonal growth and myelination in vitro

Applied Neurobiology Group, Institute of Comparative Medicine, Division of Cell Sciences, University of Glasgow Veterinary School, Glasgow, Scotland, UK.
Journal of Neuroscience Research (Impact Factor: 2.73). 12/2006; 84(8):1703-15. DOI: 10.1002/jnr.21084
Source: PubMed

ABSTRACT In vitro models of myelinating central nervous system axons have mainly been of two types, organotypic or dissociated. In organotypic cultures, the tissue fragment is thick and usually requires sectioning (physically or optically) before visual examination. In dissociated cultures, tissue is dispersed across the culture surface, making it difficult to measure the extent of myelinated fiber growth. We aimed to develop a method of culturing myelinated CNS fibers in defined medium that could be 1) studied by standard immunofluorescence microscopy (i.e., monolayer type culture), 2) used to measure axonal growth, and 3) used to evaluate the effect of substrate and media components on axonal growth and myelination. We used 120-micro m slices of embryonic murine spinal cord as a focal source of CNS tissue from which myelinated axons could extend in a virtual monolayer. Explants were cultured on both poly-L-lysine and astrocytes. The latter were used because they are the scaffold on which axonal growth and myelination occurs during normal development. Outgrowth from the explant and myelination of axons was poor on poly-L-lysine but was promoted by an astrocyte bed layer. The best myelin formation occurred in defined media based on DMEM using N2 mix; it was not promoted by Sato mix or Neurobasal medium with B27 supplement. Neuronal survival was poor in serum-containing medium. This tissue culture model should facilitate the study of factors involved in promoting outgrowth of CNS axons and their myelination. As such it is relevant to studies on myelination and spinal cord repair.

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    12/2014; 2(1):178. DOI:10.1186/s40478-014-0168-9
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    ABSTRACT: Studies of central nervous system myelination lack defined in vitro models which would effectively dissect molecular mechanisms of myelination that contain cells of the correct phenotype. Here we describe a co-culture of purified motoneurons and oligodendrocyte progenitor cells, isolated from rat embryonic spinal cord using a combination of immunopanning techniques. This model illustrates differentiation of oligodendrocyte progenitors into fully functional mature oligodendrocytes that myelinate axons. It also illustrates a contribution of axons to the rate of oligodendrocyte maturation and myelin gene expression. The defined conditions used allow molecular analysis of distinct stages of myelination and precise manipulation of inductive cues affecting axonal-oligodendrocyte interactions. This phenotypic in vitro myelination model can provide valuable insight into our understanding of demyelinating disorders, such as multiple sclerosis and traumatic diseases such as spinal cord injury where demyelination represents a contributing factor to the pathology of the disorder.
    Biomaterials 07/2014; 35(31). DOI:10.1016/j.biomaterials.2014.07.007 · 8.31 Impact Factor
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    ABSTRACT: BackgoundMyelination is a very complex process that requires the cross talk between various neural cell types. Previously, using cytosolic or membrane associated GFP tagged neurospheres, we followed the interaction of oligodendrocytes with axons using time-lapse imaging in vitro and ex vivo and demonstrated dynamic changes in cell morphology. In this study we focus on GFP tagged astrocytes differentiated from neurospheres and their interactions with axons.ResultsWe show the close interaction of astrocyte processes with axons and with oligodendrocytes in mixed mouse spinal cord cultures with formation of membrane blebs as previously seen for oligodendrocytes in the same cultures. When GFP-tagged neurospheres were transplanted into the spinal cord of the dysmyelinated shiverer mouse, confirmation of dynamic changes in cell morphology was provided and a prevalence for astrocyte differentiation compared with oligodendroglial differentiation around the injection site. Furthermore, we were able to image GFP tagged neural cells in vivo after transplantation and the cells exhibited similar membrane changes as cells visualised in vitro and ex vivo.ConclusionThese data show that astrocytes exhibit dynamic cell process movement and changes in their membrane topography as they interact with axons and oligodendrocytes during the process of myelination, with the first demonstration of bleb formation in astrocytes.
    BMC Neuroscience 05/2014; 15(1):59. DOI:10.1186/1471-2202-15-59 · 2.85 Impact Factor


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