Name: an approach to enhance alignment and myelination of dorsal root ganglion neurons
Uploaded: 2016-08-24T14:00:00
Description: Watch this Scientific Journal Video about An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons at JoVE.com

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&#39;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).

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&#160;
<ol>
	<li>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.</li>
	<li>Keep the gel mixture under vacuum for 20 min to remove air bubbles.</li>
	<li>Place the gel mixture in the ov...

<ol>
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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...

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 strongly impairs the conductive capacity of surviving axons, treatments targeting demyelination or promoting remyelination are significant for functional recovery after nerve injury 5. Thus the goal of this protocol is to illustrate an engineering approach that addresses these two issues of nerve regeneration.
Surface anisotropy, which is defined as a difference, when measured along different axes, in a material&#39;s physical or mechanical properties, has been applied to influence cell alignment, growth, and migration 6-7. In addition to topography, there are other methods to induce anisotropy. Previously, we investigated surface anisotropy induced by mechanical static pre-stretch of poly-dimethyl-siloxane (PDMS) membrane. The theory of &#34;small deformation super imposed on large&#34; predicted that the effective stiffness the cells sense in the stretched direction differs from the perpendicular direction, and this difference in effective stiffness is due to surface anisotropy 8. Mesenchymal stem cells (MSCs) cultured on a pre-stretched PDMS membrane are able to sense the anisotropy by actively pulling the surface and as a result, align in the pre-stretched direction 9. Similarly, an anisotropic surface arising from a mechanical pre-stretched surface affects the alignment, as well as growth and myelination of dorsal root ganglion (DRG) axons 10. Here we provide a protocol for inducing surface anisotropy on a static pre-stretched PDMS substrate to enhance axon regeneration 10.
To elicit axon alignment, topological features with desired patterns, reported to provide contact guidance through aligned fibers and channels 6,11-12, were demonstrated to facilitate axon alignment 11,13. However, reported techniques for inducing axon alignment through topological features, such as fibers, channels and patterning, were unable to lengthen and increase the thickness of the axons. In contrast, gradual mechanical stretching led to axon alignment in the stretch direction with longer and thicker axons that increased with the magnitude of the stretch 14. However, incorporating a powered motor device in vivo is not feasible. In contrast, static pre-stretched induced anisotropy is less complicated and can be more readily incorporated into future scaffold designs for in vivo applications.
In this protocol, a static pre-stretched cell culture system is used to induce surface anisotropy without topological features. The pre-stretched culture system is composed of a PDMS membrane, a stretchable frame and a stretching stage, whereupon the membrane is fixed onto the frame and a predetermined stretch magnitude is applied on the stretching stage. Freshly isolated DRG neurons cultured on the pre-stretched surface for up to 21 days are monitored for axon alignment and thickness. Subsequently, Schwann cells (SCs) co-cultured with the aligned axons are monitored for myelination. By incorporating pre-stretch induced surface anisotropy we were able to enhance cell alignment-differentiation and axon alignment-growth of MSCs and DRG neurons 9-10, respectively.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Polydimethylsiloxane &#65288;PDMS)</td>
			<td>Dow Corning</td>
			<td>SYLGARD 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium 1X</td>
			<td>GibcoBRL</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement 50X</td>
			<td>GibcoBRL</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax-I 100X</td>
			<td>GibcoBRL</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumax-I</td>
			<td>GibcoBRL</td>
			<td>11020-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Nerve Growth Factor-7S</td>
			<td>Invitrogen</td>
			<td>13290-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>GibcoBRL</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA/1mM EDTA</td>
			<td>GibcoBRL</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Trevigen</td>
			<td><a href="http://www.trevigen.com/item/2/7/64/220/Cultrex_PolyLLysine/">3438-100-01</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma</td>
			<td>p-6407</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoro-2 deoxy-uridine</td>
			<td>Sigma</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma</td>
			<td>U3003</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Invitrogen</td>
			<td>14170-112</td>
			<td>Isolation Buffer</td>
		</tr>
		<tr>
			<td>Type I Collagenase</td>
			<td>Worthington</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11885</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat inactivated Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH30080.03</td>
			<td></td>
		</tr>
		<tr>
			<td>BPE</td>
			<td>Clonetics</td>
			<td>CC-4009</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Calbiochem</td>
			<td>344270</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone chamber</td>
			<td>Greiner bio-one</td>
			<td>FlexiPERM ConA</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaning/etching system</td>
			<td>March Instruments</td>
			<td>PX-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Thy 1.1 antibody</td>
			<td>Sigma- Aldrich</td>
			<td>M7898</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Complement</td>
			<td>Sigma- Aldrich</td>
			<td>S-7764</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard growth medium</td>
			<td></td>
			<td></td>
			<td>For 500 ml Neurobasal Medium 1X, add 10 ml of B-27 50X, 5 ml of Glutamax-I 100X,</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>2.5 ml of Penicillin/Streptomycin (Penn/Strep), 1 ml of Albumax-I, and 1 &#956;l of NGF-- 7S (50 ug/ml).</td>
		</tr>
		<tr>
			<td>FDU and Uridine stock solution</td>
			<td></td>
			<td></td>
			<td>FDU 100mg in 10ml of ddw (10mg/ml), filter in the hood and divided in 500ul aliquots and store at -20 &#186;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Uridine 5g in 166.7ml of ddw (33mg/ml), filter in hood, divide in 200ul aliquots and store at -20 &#186;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Take 61.5ul of FDU (10mg/ml) and 20.5ul of Uridine(33mg/ml), and add 4918ul of ddw to a final stock concentration,</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>then divide in 1 ml aliquots and store at -20 &#186;C.</td>
		</tr>
	</tbody>
</table>

an approach to enhance alignment and myelination of dorsal root ganglion neurons

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.

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 &#946;-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...

Watch this Scientific Journal Video about An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons at JoVE.com

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

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 ...

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