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.

  • Source
    • "Cell-culture methods Cell-lines were grown in Dulbecco's Modified Eagle's Medium (DMEM; Gibco) supplemented with 10% Fetal Bovine Serum (FBS; Gibco). Dissociated spinal cord cells were prepared from mice embryos at 13.5 day of gestation using a modified procedure originally described for explant cultures (Thomson et al. 2006). Meninges-free spinal cord tissue was mechanically minced, incubated with trypsin and collagenase at 37°C, and the reaction was terminated with soybean trypsin inhibitor solution (Leibovitz's L15 medium with 0.52mg/ml soybean trypsin inhibitor, 0.04mg/ml bovine pancreas DNAse, 3mg/ml bovine serum albumin). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Oligodendrocytes form an insulating multilamellar structure of compact myelin around axons, which allows efficient and rapid propagation of action potentials. However, little is known about the molecular mechanisms operating at the onset of myelination and during maintenance of the myelin sheath in the adult. Here we use a genetic cell ablation approach combined with Affymetrix GeneChip microarrays to identify a number of oligodendrocyte-enriched genes that may play a key role in myelination. One of the "oligogenes" we cloned using this approach is Tmem10/Opalin, which encodes for a novel transmembrane glycoprotein. In situ hybridization and RT-PCR analysis revealed that Tmem10 is selectively expressed by oligodendrocytes and that its expression is induced during their differentiation. Developmental immunofluorescence analysis demonstrated that Tmem10 starts to be expressed in the white matter tracks of the cerebellum and the corpus callosum at the onset of myelination after the appearance of other myelin genes such as MBP. In contrast to the spinal cord and brain, Tmem10 was not detected in myelinating Schwann cells, indicating that it is a CNS-specific myelin protein. In mature oligodendrocytes, Tmem10 was present at the cell soma and processes, as well as along myelinated internodes, where it was occasionally concentrated at the paranodes. In myelinating spinal cord cultures, Tmem10 was detected in MBP-positive cellular processes that were aligned with underlying axons before myelination commenced. These results suggest a possible role of Tmem10 in oligodendrocyte differentiation and CNS myelination.
    Glia 08/2008; 56(11):1176-86. DOI:10.1002/glia.20688 · 6.03 Impact Factor
  • Source
    • "3E,F, arrows). Our observations are in agreement with Thomson et al. (2006), who used astrocytes derived from neurospheres as a monolayer for spinal cord explants to study myelination. These data, therefore, suggest that astrocytes derived from neurospheres and astrocytes isolated from the cortex have a similar cellular composition, antigenic phenotype, and morphology. "
    [Show abstract] [Hide abstract]
    ABSTRACT: We have examined the interaction between olfactory ensheathing cells (OECs), Schwann cells (SC), oligodendrocytes, and CNS axons using cultures generated from embryonic rat spinal cord. Oligodendrocyte process extension and myelination in these cultures was poor if the cells were plated on OECs or SCs. Myelin internodes and nodes of Ranvier formed frequently if these cultures were plated onto monolayers of neurosphere-derived astrocytes (NsAs). In the myelinated fibers generated on NsAs, Nav channels, caspr, and neurofascin molecules were correctly assembled at the nodes of Ranvier. The density of neurites, survival, and antigenic differentiation of oligodendrocytes was similar on OEC and NsAs monolayers. However, on OEC monolayers, despite a transient increase in the number of endogenous oligodendrocytes, there was a decrease in oligodendrocyte process extension and axonal ensheathment when compared with cultures plated on NsAs monolayers. To determine if these changes were due to axonal or glial factors, spinal cord oligodendrocytes were plated onto monolayers of OECs, NsAs, and poly-L-lysine in the absence of neurons. In these cultures, process extension and myelin-like membrane formation by oligodendrocytes was improved on monolayers of OEC. This suggests that inhibition of process extension is mediated via cross-talk between OECs and neurites. In cultures containing axons plated on OEC monolayers, oligodendrocyte process formation, axonal ensheathment, and myelination occurred albeit lower if the cultures were supplemented with NsAs conditioned medium. These data suggest OECs can permit neurite extension and oligodendrocyte proliferation, but lack secreted factor(s) and possible cell-cell contact that is necessary for oligodendrocyte process extension and myelination.
    Glia 05/2008; 56(7):750-63. DOI:10.1002/glia.20650 · 6.03 Impact Factor
  • Source
    • "Astrocytes were prepared by differentiating neural stem cells using a locally modified version of the methods described by Reynolds and Weiss [18] and Zhang et al. [19]. The seeding of structures required a large number of cells, which was easier to achieve by differentiating astrocytes from neurospheres compared to the isolation and culture of primary astrocytes [20]. We have shown recently that these cells are indeed comparable in function as axonal density and their subsequent myelination were similar if spinal cord neurons were plated onto either cell type (Sørensen et al., submitted to Glia). "
    [Show abstract] [Hide abstract]
    ABSTRACT: After spinal cord injury neuronal connections are not easily re-established. Success has been hampered by the lack of orientation of neurites inside scar tissue and a lack of neurites crossing out of the site of injury. Oriented scaffolds in biodegradable polymers could be an excellent way to support both the orientation of neurites within the injury site as well as aiding their crossing out of the lesion. To establish the validity of using grooved micro-topography in polycaprolactone in combination with glia we have studied the long-term (3 weeks) orientation of neuronal cells on monolayers of astrocytes on the top of grooved topographies of various dimensions. We find that neurites are significantly aligned by groove/ridge type topographies which are "buried" under a monolayer of astrocytes for up to 3 weeks. This alignment is significantly lower than that of neurites growing directly on the topography, but these neurons do not survive on the poly-l-lysine coated polymer for more than a week. The alignment of neurites on the astrocyte layer to the underlying topography decreases over time, and with groove width. Topographies with 12.5 or 25 microm lateral dimension appear optimal for the long-term alignment and can support myelination. We have shown for the first time that micro-topography can act through an overlaid astrocyte layer and results in aligned neurites in long-term culture and that these can be myelinated by endogenous oligodendrocytes.
    Biomaterials 01/2008; 28(36):5498-508. DOI:10.1016/j.biomaterials.2007.08.034 · 8.31 Impact Factor
Show more


1 Download