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 oven for overnight curing at 60 &#176;C.</li>
	<li>After the PDMS membrane cures, treat the surface with oxygen plasma using a plasma cleaning/etching system for 3 min at 165 mTorr and 65 sccm flow of O2.</li>
	<li>Cut a piece of rectangular membrane (5&#160;&#215; 3.5 cm) from the dish, while being cognizant which side is oxygen-treated, make sure the oxygen-treated side is facing up and fix onto a stretching frame. Place the frame on a stretching stage and evenly stretch by turning the knob on the stage until it reaches 10% elongation (or some other pre-determined stretch; the elongation can be directly read from the stretching stage) in the longer axis 9. Fix the stretch by tightening the screws on the frame.</li>
	<li>Remove the frame from the stage. Ensure the membrane surface is dry and free of dust, then place a silicone chamber onto the membrane allowing the sticky side of the chamber to attach tightly to the membrane. 
	Note: The silicone chamber provides a well for retaining the cell medium on the PDMS membrane. The device design is shown in Figure 1.</li>
	<li>Sterilize the surface with UV for 10 min.</li>
	<li>Add 1 ml Poly-L-Lysine (PLL) (0.01% in Phosphate Buffered Saline) to the PDMS surface within the chamber, within 6 hr of plasma treatment and incubate for 2 hr at 37 &#176;C prior to seeding the DRG neurons. This enhances cell attachment.</li>
</ol>
2. Isolation of DRG
<ol>
	<li>Prepare standard growth medium for the DRG neurons.</li>
	<li>Prior to the isolation, autoclave the isolation equipment and filter reagents. Add 5 ml of isolation buffer into one well of a 6-well plate, and place the plate on ice. Prepare 10 ml of dissociation medium by adding 1 ml of collagenase A (500 U/ml) to 9 ml of 0.05% Trypsin-EDTA (1 mg/ml), filter the solutions with a 0.22 &#181;m filter and place on ice.</li>
	<li>Use ten to twelve 5 - 7 days&#39; old Sprague-Dawley rats for the isolation. Spray the pups with 75% isopropanol, and sacrifice by decapitation.</li>
	<li>Cut away the skin overlying the spinal cord from the back and remove any excess tissue around the spine. Under a surgical magnifier with the surgical spot lights on, make the first incision from the neck and then one cut along the spine on both sides with scissors. Detach the spine from the body of the pups and remove the excess muscles. Then, use scissors to cut along the long axis of the spine and use tweezers to open the spine completely and extract the spinal cord.Remove the tip from a pipette to create a larger opening for transferring the DRGs with isolation buffer into a sterile 15 ml tube.</li>
	<li>Using a fine pair of tweezers, remove the ganglion from the bone pocket and collect approximately 10 &#8211; 16 DRG from both sides. Trim the nerve roots and transfer the ganglia to the ice-cold isolation buffer.</li>
	<li>Remove the tip from a pipette to create a larger opening for transferring the DRGs with isolation buffer into a sterile 15 ml tube. After the chemical dissociation, centrifuge the ganglia at 900 x g and 4 &#176;C for 5 min.</li>
	<li>Let the tissues settle to the bottom of the tube and gently remove the isolation buffer on top and add 10 ml of dissociation medium into the tube.</li>
	<li>Incubate the dissected tissues in the dissociation medium in a 37 &#176;C water bath for 1 hr while shaking the tube every 5 - 10 min.</li>
	<li>After the chemical dissociation, centrifuge the ganglia at 900 x g and 4 &#176;C for 5 min.</li>
	<li>Remove the supernatant and re-suspend the pellet in 10 ml standard growth media and vortex.</li>
	<li>Centrifuge the dissociated cells as indicated in section 2.9 once again.</li>
	<li>Remove the supernatant, re-suspend the pellet in 12 ml of standard growth media and vortex.</li>
</ol>
3. Culture of DRG on Pre-stretched Surface
<ol>
	<li>Prior to seeding the cells, remove the PLL solutions from the chamber and rinse the PDMS surface with sterile water and air dry.</li>
	<li>After re-suspending the cells, let the suspension set for 1 - 2 min to allow the debris to settle to the bottom of the tube. Add 1.5 ml of cell suspension into each stretch chamber and incubate at 37 &#176;C at 5% CO2.</li>
	<li>To eliminate glial cells, at 1 day in vitro (DIV), add 10 &#181;l (or 15 &#181;l) mixture of fluoro-2 deoxy-uridine and uridine (FDU-U) stock solution (See Table of Materials) to each well. After 7 hr, replace this medium with fresh standard growth medium and place the pre-stretched culture device in a 37 &#176;C incubator with 5% CO2.</li>
	<li>During the cell culture period (2 - 3 weeks), change the media every two days by replacing half the spent medium with fresh medium. Note: After culturing on the stretched and unstretched surfaces for 2 weeks, purified SCs are added to the chamber and cultured for another week (step 4.7).</li>
</ol>
4. Co-culture of Schwann Cells (SCs) with DRG Neurons on Pre-stretched Surface
<ol>
	<li>Isolate SCs as described by Dr. Campana&#39;s group previously 15.
	<ol>
		<li>Briefly, isolate the SCs from a 1 day old pup. Collect and dissociate the sciatic nerves both chemically (Trypsin-EDTA and Collagenase A) and mechanically (18 gauge needle/10 ml syringe) 15.</li>
		<li>Seed the dissociated cells into poly-D-lysine (PDL) coated T25 flask and culture at 37 &#176;C at 5% CO2. On day 5, purify the cells through antibody selection using Anti-thy 1.1 antibody and Rabbit Complement and subculture the purified cells to passage 2 - 4. Use culture medium containing Dulbecco&#39;s Modified Eagle Medium (DMEM), 10% Fetal bovine serum (FBS), 1% Penicillin/Streptomycin (Penn/Strep), 21 &#181;g/ml Bovine Pituitary Extract (BPE), and 4 &#181;M Forskolin.</li>
	</ol>
	</li>
	<li>Remove the culture medium from the SCs, and add 5 ml 0.05% Trypsin-EDTA into the flask. Incubate the cells at 37 &#176;C at 5% CO2 for 2 - 3 min.</li>
	<li>Check cells under the optical microscope using a 10X objective to see if they lift up from the flask, then add 5 ml of culture medium and mix with the cells in the flask.</li>
	<li>Add the cell suspension to a 15 ml centrifuge tube and centrifuge at 200 x g and 20 &#176;C for 5 min.</li>
	<li>Remove the supernatant and resuspend the pellet in 7 ml standard DRG growth media.</li>
	<li>Take 10 &#181;l of cell suspension into a 0.5 ml microcentrifuge tube, mix with 10 &#181;l of trypan blue, and then count the cell number using a hemocytometer under optical microscopy using a 10X objective. Dilute the cell suspension to 5,000 cells per ml by adding DRG growth media.</li>
	<li>Remove 0.5 ml of medium from the DRG culture in the stretched chamber and add 0.5 ml of SC suspension to the DRG culture.</li>
	<li>Culture the cells for 1 week at 37 &#176;C and 5% CO2. Change the media every two days by replacing half the spent medium with fresh standard growth medium for DRG. After 1 week co-culture, process the cells by immunohistochemistry 10.</li>
</ol>

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The authors do not have any conflict of interest to disclose.

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) after plasma treatment attenuates over time. Since the plasma treatment is applied to one side of the membrane, care must be taken after peeling the PDMS off the dish to ensure the correct side is placed on the frame, that is, the plasma treated surface should be facing up for the cells to attach to.
The DRGs are located inside the bone pockets that align alongside the spine. Since the color of the vertebra is white in young pups, it is difficult to distinguish the DRGs from the vertebra. In this case, it is important to place the spine close to the spot light. Under the light, the DRGs turn transparent while the bone remains white and opaque. It is important to keep the dissection buffer on ice and at a physiological pH, since the pH influences the viability of the neural cells. The isolated tissues are very viscous and tend to stick to the pipette tip when transferring to a centrifuge tube. Thus cutting the very tip of the pipette to generate a larger opening facilitates the transfer.
In some protocols, there is one more centrifugation step required with the isolated tissues before adding the dissociation buffer. In lieu of this step, our protocol requires the tissues to settle in the dissection buffer for a couple of minutes, which reduces the extra centrifugation step that otherwise would impact the viability and density of the cells. The cell density is also dependent on the number of pups sacrificed, and thus is important to maintain a consistent number of pups each time. There are also glial cells that are isolated along with the DRG neurons, thus a mitotic inhibitor, FDU-U, is added to the culture medium at 1 DIV to suppress the growth of the glial cells. This is commonly used to inhibit glial cells during the early growth period of the neurons, although some protocols prefer a combination of arabinofuranosylcytidine (AraC) and FDU-U. The molar concentration of FDU-U added to the culture varies in the different protocols, depending on the density of the isolated cells. We found the mitotic inhibitor can be added multiple times (at the concentration specified in step 3.3), as needed (if glial cells are present), during the entire DRG culture. It is generally not recommended to add a large amount (more than 15 &#181;l) at any given time since it impacts the attachment of the DRG neurons.
Plasma treatment enhances surface hydrophilicity and the PLL coating provides a charged surface for axons to initiate attachment. According to references in the literature, the plasma treatment induced hydrophilicity will attenuate over time, under dry conditions 16-17. However PDMS will undergo a slow reaction with water under liquid conditions and will gradually change from being hydrophobic to hydrophilic 18. Therefore even though the plasma treatment attenuates over time, it should not impact our results. For the PLL coating, according to the product information from the supplier, it is stable over years if placed under favorable conditions. Once the cells attach onto the PLL coating, they generally will not detach. Additionally, the PLL coating helps with the initial attachment, and as the cells grow they secrete Extracellular matrix (ECM) proteins which further help them to remain anchored to the surface 19. Future design improvements could include imbedding peptides into the PDMS network during crosslinking, which would eliminate the plasma treatment and PLL coating steps.
The protocol, herein, described an engineering approach that generated surface anisotropy to successfully induce axon alignment, growth and myelination. The approach can be experimentally expanded in two ways. First, the anisotropic surface could be applied to create nerve tissues in culture, and the in vitro nerve tracts could then be transplanted in vivo. Second, the concept of surface anisotropy could be incorporated into the design of transplantable scaffolds that could be used in vivo to enhance the alignment of the regenerating axons from the injury site and myelination from the host SCs. Compared to other approaches which require multiple and complicated devices 20-22, as well as the addition of growth factors, our platform is relatively simple and easy to apply, and relies only on pre-stretch to enhance both axon alignment and myelination without the need for growth factor addition. Furthermore we have tested this concept of pre-stretch in the experimental design on both MSCs and DRG neurons which could easily be applied to other cell types. The current design was used to investigate axon growth on 2-dimensional surfaces, however in future the device could be upgraded to enable the testing of 3-dimenstional cell cultures. Future applications in nerve repair and transplantation could incorporate pre-stretch into the transplantable scaffolds made from biodegradable natural or synthetic materials containing crosslinked peptides along with controlled-release drug properties.

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 interconnected network on the unstretched PDMS substrate.
In addition to inducing axon alignment, pre-stretch induced anisotropy enhanced the fasciculation of axons. The aligned axons on the stretched surface formed axonal bundles, which differed from the connected axonal network observed on the unstretched surface. These fascicular bundles on the pre-stretched surface resembled nervous tissue tracts in vivo. Figure 3 shows axons of the DRG neurons on pre-stretched and unstretched surfaces after 21 days of culture. As Figure 3 illustrates, the axons on the pre-stretched substrate aggregated together to form bundles, which appeared similar to fascicular tracts. This suggested the pre-stretched surface was able to promote extensive axon growth in the direction of the pre-stretch, which are important in enhancing regeneration of nervous tissue.
To assess myelination, SCs were co-cultured with the aligned axons. After culturing the DRG neurons on the stretched and unstretched surfaces for 2 weeks, purified SCs were added to the chamber and cultured for another week. In Figure 4, the axons of the co-culture are stained with &#946;-III-tubulin (green), and the SCs are stained with P0 (red). P0 is a marker of mature SCs and is also a component of the myelin sheath, as well as considered an indicator of myelination. There is significant co-localization of green and red on the pre-stretched surface, suggestive of SCs attaching to the axons on the stretched surface, but not on the unstretched surface where the green and red stains are randomly distributed. The pre-stretched surfaces enhanced axon alignment, fascicular-like tract formation, and attachment of SCs to the regenerated axons, all critical for myelination.
<img alt="Figure 1" src="/files/ftp_upload/54085/54085fig1.jpg" /> 
Figure 1: Schematic for the Pre-stretch Procedure. (a) A piece of PDMS membrane is clipped onto two strips of the frame that are slidable. The frame of the device is approximately 7&#34; x 5&#34;; the braces are about 5&#34; x 1; the clamps on the braces are about 3&#34; x .25&#34;, and the cup is about 1.5&#34; diameter wide open top, 1&#34; open bottom, and about 0.25&#34; tall. (b) Place the frame onto the stretching stage, turn the roller to apply 10% elongation, and then fix the positions of the strips by tightening the screws. (c) Remove the frame from the stretching stage, and attach a silicon chamber onto the membrane. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54085/54085fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54085/54085fig2.jpg" /> 
Figure 2: Axon Alignment on Pre-stretched and Unstretched Surface. Fluorescent images of DRG neurons seeded on the (a) static pre-stretched surface for 12 days, compared with DRG cells seeded on the (b) unstretched surface for 12 days. Axons aligned in the pre-stretched direction, while axons aligned randomly on the unstretched control surface. Axons were stained with anti-mouse &#946;-III tubulin primary antibody, and imaged with confocal microscope using a 10X objective. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54085/54085fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54085/54085fig3.jpg" /> 
Figure 3: Axon Thickness Growth on Stretched vs. Unstretched PDMS Substrate. Phase contrast images of DRG axons on (a) stretched and (b) unstretched surfaces after 21 days. Axons on the stretched surface formed thicker bundles, while on the unstretched surface the axons formed inter-connected axonal network. Images were taken using inversed phase contrast microscope using a 10X objective. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54085/54085fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54085/54085fig4.jpg" /> 
Figure 4: Co-cultured SCs with Aligned DRG Axons on Stretched vs. Unstretched PDMS Substrates. After culturing on the stretched and unstretched surfaces for 2 weeks, purified SCs were added to the chamber and cultured for another week. (a) &#946;-III-tubulin (green) staining for axons on the stretched surface; (b) Overlay of &#946;-III-tubulin (green), P0 (red) staining on the stretched surface; (c) &#946;-III-tubulin (green) staining for axons on the unstretched surface; (d) Overlay of &#946;-III-tubulin (green), P0 (red) staining on the unstretched surface. On the pre-stretched surface, the red and green stains colocalize, suggesting the SCs are attached to and likely myelinating the axons. On the unstretched surface, the red and green stains are randomly distributed, suggesting the SCs are not attached to the axons. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54085/54085fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

an-approach-to-enhance-alignment-myelination-dorsal-root-ganglion

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.4K Views.  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.

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

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...