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Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Neuroscience

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Published: August 24th, 2016

DOI:

10.3791/54085

1Department of Chemical Engineering and Materials Science, Michigan State University, 2Department of Biochemistry and Molecular Biology, Michigan State University

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

In nerve injuries, the proximal and distal nerve stumps are often prevented from direct realignment of nerve fascicles due to the lesion site 1-2. Normally, axon tracts are composed of highly ordered and aligned bundles of axons, which form complex networks of connectivity. However, nerve regeneration is a chaotic process due to poorly organized axon alignment 3-4. Therefore, to generate a sufficient number of regenerating axons that bridge the lesion site, it is necessary to induce well organized axonal alignment. Additionally, demyelination accompanies nerve injuries due to death of the myelinating cells at the injury site. Since demyelination ....

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All procedures for the isolation of the cells were approved by the Institutional Animal Care and Use Committee at Michigan State University.

1. Preparation of Pre-stretched Anisotropic Surface 

  1. Mix a 10:1 solution of base and curing agent and pour the mixture into a tissue culture dish (12 cm diameter). Use 4,900 mg base and 490 mg curing agent for the total crosslinking mixture.
  2. Keep the gel mixture under vacuum for 20 min to remove air bubbles.
  3. Place the gel mixture in the ov.......

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The pre-stretched cell culture system promoted DRG axon alignment 10. DRG neurons were cultured onto pre-stretched and unstretched surfaces for 12 days. The axons were stained for β-III-tubulin to demonstrate their alignment. Figure 2 compares axon orientation on the pre-stretched and unstretched PDMS substrates after 12 days of culture. The DRG axons aligned parallel to the stretched direction, whereas they showed random alignment and formed an interconne.......

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To induce axon alignment on pre-stretched surface, there are two critical steps: 1) the PDMS membrane must be flat and of homogenous thickness; and 2) glial cells must be removed from the DRG. After mixing the PDMS and crosslinker and curing in an oven, the crosslinked PDMS gel should be kept on a flat bench top and handled carefully to avoid any tilting. The oxygen plasma treatment of the PDMS membrane should be followed within 6 hr by PLL coating, since the hydrophilicity of the surface (required for cell attachment) a.......

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The authors would like to thank Eric Vasco for his assistance in the preparation of the PDMS substrates, Dr. Shiyong Wang in Dr. Marina Mata's lab at University of Michigan for helpful suggestions and training of the DRG isolation, and Dr. Mark Tuszynski and Dr. W. Marie Campana at UC San Diego for helpful suggestions and protocol for the SC isolation. This study was supported in part by the National Science Foundation (CBET 0941055 and CBET 1510895), the National Institute of Health (R21CA176854, R01GM089866, and R01EB014986).

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Name Company Catalog Number Comments
Polydimethylsiloxane (PDMS) Dow Corning SYLGARD 184
Neurobasal Medium 1X GibcoBRL 21103-049
B27 Supplement 50X GibcoBRL 17504-044
Glutamax-I 100X GibcoBRL 35050-061
Albumax-I GibcoBRL 11020-021
Nerve Growth Factor-7S Invitrogen 13290-010
Penicillin-streptomycin GibcoBRL 15140-122
0.05% Trypsin-EDTA/1mM EDTA GibcoBRL 25300-054
Poly-L-Lysine Trevigen 3438-100-01
Poly-D-Lysine Sigma p-6407
Fluoro-2 deoxy-uridine Sigma F0503
Uridine Sigma U3003
Hank’s Balanced Salt Solution (HBSS) Invitrogen 14170-112 Isolation Buffer
Type I Collagenase Worthington LS004196
DMEM Gibco 11885
Heat inactivated Fetal Bovine Serum Hyclone SH30080.03
BPE Clonetics CC-4009
Forskolin Calbiochem 344270
Silicone chamber Greiner bio-one FlexiPERM ConA
Plasma cleaning/etching system March Instruments PX-250
Anti-Thy 1.1 antibody Sigma- Aldrich M7898
Rabbit Complement Sigma- Aldrich S-7764
Standard growth medium For 500 ml Neurobasal Medium 1X, add 10 ml of B-27 50X, 5 ml of Glutamax-I 100X,
2.5 ml of Penicillin/Streptomycin (Penn/Strep), 1 ml of Albumax-I, and 1 μl of NGF-- 7S (50 ug/ml).
FDU and Uridine stock solution FDU 100mg in 10ml of ddw (10mg/ml), filter in the hood and divided in 500ul aliquots and store at -20 ºC.
Uridine 5g in 166.7ml of ddw (33mg/ml), filter in hood, divide in 200ul aliquots and store at -20 ºC.
Take 61.5ul of FDU (10mg/ml) and 20.5ul of Uridine(33mg/ml), and add 4918ul of ddw to a final stock concentration,
then divide in 1 ml aliquots and store at -20 ºC.

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