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: 2.59). 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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    • "Myelinating cultures were generated from E13.5 (embryonic day) pups as described previously [24,45,46]. Briefly the spinal cord was dissected, the meninges removed, and minced using a scalpel blade prior to enzymatic dissociation (100 μl of 2.5% trypsin, Invitrogen, Paisley, UK and 100 μl of 1.33% collagenase, ICN Pharmaceuticals, UK). "
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    ABSTRACT: Backgound Myelination 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. Results We 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. Conclusion These 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 · 2.67 Impact Factor
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    • "There have been numerous studies on explanted nerves and peripheral nerve recovery in vitro from the proximal side but only a few are on the distal side [15]–[22]. In the present study, we observed growing axons from the distal side of an injured peripheral nerve in Gelfoam® histoculture. "
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    ABSTRACT: We have previously reported that hair follicles contain multipotent stem cells which express nestin. The nestin-expressing cells form the hair follicle sensory nerve. In vitro, the nestin-expressing hair follicle cells can differentiate into neurons, Schwann cells, and other cell types. In the present study, the sciatic nerve was excised from transgenic mice in which the nestin promoter drives green fluorescent protein (ND-GFP mice). The ND-GFP cells of the sciatic nerve were also found to be multipotent as the ND-GFP cells in the hair follicle. When the ND-GFP cells in the mouse sciatic nerve cultured on Gelfoam® and were imaged by confocal microscopy, they were observed forming fibers extending the nerve. The fibers consisted of ND-GFP-expressing spindle cells, which co-expressed the neuron marker β-III tubulin, the immature Schwann-cell marker p75(NTR) and TrkB which is associated with neurons. The fibers also contain nestin-negative spherical cells expressing GFAP, a Schwann-cell marker. The β-III tubulin-positive fibers had growth cones on their tips expressing F-actin, indicating they are growing axons. When the sciatic nerve from mice ubiquitously expressing red fluorescent protein (RFP) was co-cultured on Gelfoam® with the sciatic nerve from ND-GFP transgenic mice, the interaction of nerves was observed. Proliferating nestin-expressing cells in the injured sciatic nerve were also observed in vivo. Nestin-expressing cells were also observed in posterior nerves but not in the spinal cord itself, when placed in 3-D Gelfoam® culture. The results of the present report suggest a critical function of nestin-expressing cells in peripheral nerve growth and regeneration.
    PLoS ONE 06/2013; 8(6):e67153. DOI:10.1371/journal.pone.0067153 · 3.23 Impact Factor
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    • "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). "
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    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
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