Name: transplantation of schwann cells inside pvdf-trfe conduits to bridge transected rat spinal cord stumps to promote axon regeneration across the gap
Uploaded: 2017-11-03T12:30:00
Description: Watch this Scientific Journal Video about Transplantation of Schwann Cells Inside PVDF-TrFE Conduits to Bridge Transected Rat Spinal Cord Stumps to Promote Axon Regeneration Across the Gap at JoVE.com

We would like to thank the Viral Vector and Animal Cores at the Miami Project to Cure Paralysis for producing the lenti-GFP-virus and providing animal care, respectively, and the Histology and Imaging Cores for the use of the cryostat, confocal microscope, and fluorescent microscope with Stereo Investigator. Funding was provided by NINDS (09923), DOD (W81XWH-14-1-0482), and NSF (DMR-1006510). M.B. Bunge is the Christine E Lynn Distinguished Professor of Neuroscience.

Female adult Fischer rats (180 - 200&#160;g body weight) are housed according to NIH and USDA guidelines. The institutional Animal Care and Use Committee (IACUC) of the University of Miami approved all animal procedures.
1. Pre-Transplantation Preparation
<ol>
	<li>Conduit preparation.
	<ol>
		<li>Cut the conduit to 5 mm in length using a #10 blade under a dissecting microscope. 
		NOTE: The inner diameter of the conduit is between 2.4-2.7 mm; the outer diameter is betw...

The most critical step in creating an effective transection model is severing the spinal cord in one or two cuts. A 2-2.5 mm gap between the rostral and caudal spinal cord stumps should be present at the transection site. The three most likely reasons for such a gap not appearing are (1) the dorsal/ventral roots were not removed properly, (2) the ventral dura was not removed adequately, and/or (3) the animal was not positioned properly on the roll placed beneath her.
To perform an effective co...

Spinal cord injury repair is a complex and challenging problem that will require a combinatorial treatment strategy involving, for example, the use of cells and a biomaterial to provide a favorable microenvironment for transplanted cell function and axon regeneration at the site of injury. Hemisection1,2,3,4,5,6,7,8,9 and complete transection10,11,12,13,14,15,16,17,18,19,20,21,22 models are frequently used to assess the effects of biomaterial-based bridging therapies. The advantage of using a hemisection model is that it provides more stability for the bridging construct compared to complete transection. However, in hemisection models, it is difficult to prove axon regeneration as an outcome of the applied therapeutic method due to the presence of spared tissue. The complete transection model is the most rigorous method to demonstrate axon regeneration.
Various natural and synthetic materials have been studied for use as an injectable gel, a pre-formed gel placed in contusion or hemisection models, or as a structured conduit into hemisection or complete transection models (detailed in the reviews23,24,25). In situ gelling of an injectable matrix/SC mixture creates a more permissive interface between the transplant and the host cord for axon crossing26,27 compared to pre-gelled matrix/SC implants5,18,19,28. In situ gelling allowed the matrix to contour around the irregular host interfaces whereas a more rigid and structured conduit or a less moldable pre-formed gel could not. A structured conduit often provides contact guidance and implant stability in contrast to an injectable matrix. The protocols presented here describe a surgical procedure that takes advantage of both an injectable basement membrane matrix (e.g., matrigel, see the Table of Materials, referred to as injectable matrix here) and a structured conduit to evaluate axon regeneration in the most rigorous spinal cord injury model.
Electrospun poly-vinylidenedifluoride-trifluoroethylene (PVDF-TrFE) aligned fibrous hollow conduits are used in our experimental approach. PVDF-TrFE is a piezoelectric polymer that generates a transient charge when mechanically deformed and has been shown to promote neurite extension and axon regeneration both in vitro29,30 and in vivo31. Electrospinning is a common scaffold fabrication method that can rapidly produce reliable fibrous scaffolds using a variety of polymers with controllable properties such as fiber alignment, fiber diameter, and thickness of the scaffold for neural and other applications32,33,34.
Numerous studies of rat SCs transplanted into spinal cord injury sites have demonstrated treatment efficacy5,9,18,19,20,21,26. These transplants are neuroprotective for tissue surrounding the lesion, reduce lesion cavity size, and promote axon regeneration into the lesion/transplant site and myelination of the regenerated axons. Human SCs can be autologously transplanted, an advantage when compared to most other neural-related cells24. After a peripheral nerve biopsy, SCs can be isolated and purified and will proliferate to the desired amount for transplantation into humans. Autologous SC transplantation for spinal cord injured patients has been proven to be safe in Iran35,36,37,38, China39,40, and the United States41,42. SCs are known to secrete numerous neurotrophic factors and extracellular matrix proteins important for axon growth and to play an essential role in axon regeneration after peripheral nerve injury. Our goal here is to describe methods which can investigate conduit designs to improve the outcome of SC transplantation in a complete rat spinal cord transection model.

<table>
	<tbody>
		<tr>
			<td>Cryogenic vials</td>
			<td>ThermoFisher Scientific</td>
			<td>5000-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Petri dish</td>
			<td>VWR</td>
			<td>25382-428</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium: nutrient mixture F-12</td>
			<td>ThermoFisher Scientific</td>
			<td>11039-021</td>
			<td>&#34;DMEM/F12&#34; in protocol.</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140-122</td>
			<td>&#34;Pen/Strep&#34; in protcol.</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH300-70-03</td>
			<td>&#34;FBS&#34; in protocol.</td>
		</tr>
		<tr>
			<td>Pituitary extract</td>
			<td>Biomedical Technologies</td>
			<td>BT-215</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma-Aldrich</td>
			<td>F6886</td>
			<td></td>
		</tr>
		<tr>
			<td>Heregulin</td>
			<td>R&#38;D Systems</td>
			<td>396-HB/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P2636</td>
			<td>&#34;PLL&#34; in protocol.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium</td>
			<td>ThermoFisher Scientific</td>
			<td>11965-092</td>
			<td>&#34;DMEM&#34; in protocol.</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution</td>
			<td>ThermoFisher Scientific</td>
			<td>14170-112</td>
			<td>&#34;HBSS&#34; in protocol.</td>
		</tr>
		<tr>
			<td>Tryspin-EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Female Fischer rat (160-180g)</td>
			<td>Envigo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas scissor, straight</td>
			<td>FST</td>
			<td>15018-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Vedco Inc</td>
			<td>5098976106</td>
			<td>100 mg/ml</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Lloyd Inc</td>
			<td>AnaSed</td>
			<td>20 mg/ml</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>APP Pharmaceuticals</td>
			<td>NDC 63323-010-02</td>
			<td>Can be any brand of choice.</td>
		</tr>
		<tr>
			<td>Micro Spatula</td>
			<td>FST</td>
			<td>10089-11</td>
			<td>Can be any brand of choice.</td>
		</tr>
		<tr>
			<td>Curved scissors with blunt end</td>
			<td>FST</td>
			<td>14017-18</td>
			<td>Can be any brand of choice.</td>
		</tr>
		<tr>
			<td>Blunt forceps</td>
			<td>FST</td>
			<td>11006-12</td>
			<td>Can be any brand of choice.</td>
		</tr>
		<tr>
			<td>rongeur</td>
			<td>FST</td>
			<td>16121-14</td>
			<td>Can be any brand of choice.</td>
		</tr>
		<tr>
			<td>Angled spring scissors</td>
			<td>FST</td>
			<td>15006-09</td>
			<td>Can be any brand of choice.</td>
		</tr>
		<tr>
			<td>Absorption triangles</td>
			<td>FST</td>
			<td>18105-03</td>
			<td>Can be any brand of choice.</td>
		</tr>
		<tr>
			<td>Gelfoam</td>
			<td>Henry Schein</td>
			<td>9083300</td>
			<td>&#34;Compressed foam&#34; in protocol.</td>
		</tr>
		<tr>
			<td>#10 blades</td>
			<td>Sklar</td>
			<td>06-3010</td>
			<td>Can be any brand of choice.</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td>&#34;Injectable matrix&#34; in protocol.</td>
		</tr>
		<tr>
			<td>Chicken anti-green fluorescent protein antibody</td>
			<td>Millipore</td>
			<td>AB16901</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse RT97 hybridoma antibody</td>
			<td>DSHB</td>
			<td>RT97</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-neurofilament antibody</td>
			<td>Encor Biotechnology, Inc</td>
			<td>PRCA-NF-H</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal Rabbit anti-Glial Fibrillary Acidic Protein antibody</td>
			<td>Dako</td>
			<td>Z033401</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-chicken IgG (H+L)</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11039</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 goat anti-rabbit IgG (H+L)</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11035</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 goat anti-rabbit IgG (H+L)</td>
			<td>ThermoFisher Scientific</td>
			<td>A-21244</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 goat anti-mouse IgG (H+L)</td>
			<td>ThermoFisher Scientific</td>
			<td>A-21236</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscopy</td>
			<td>Nikon</td>
			<td>clsi</td>
			<td></td>
		</tr>
	</tbody>
</table>

transplantation of schwann cells inside pvdf-trfe conduits to bridge transected rat spinal cord stumps to promote axon regeneration across the gap

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord injury. The complete transection model, although not as clinically relevant as the contusion model, is the most rigorous method to evaluate axon regeneration. In the contusion model, it is difficult to distinguish regenerated from sprouted or spared axons due to the presence of remaining tissue post injury. In the complete transection model, a bridging method is necessary to fill the gap and create continuity from the rostral to the caudal stumps in order to evaluate the effectiveness of the treatments. A reliable bridging surgery is essential to test outcome measures by reducing the variability due to the surgical method. The protocols described here are used to prepare Schwann cells (SCs) and conduits prior to transplantation, complete transection of the spinal cord at thoracic level 8 (T8), insert the conduit, and transplant SCs into the conduit. This approach also uses in situ gelling of an injectable basement membrane matrix with SC transplantation that allows improved axon growth across the rostral and caudal interfaces with the host tissue.

The goal of using this surgical technique is to evaluate the use of a structured conduit and injectable matrix that maximizes SC function after transplantation into completed transected spinal cords. Three weeks after transplantation, the animals are perfused with 4% paraformaldehyde and the spinal columns are grossly dissected and fixed in the same fixative for another 24 h. The spinal cord is then dissected and the samples for cryostat sagittal sections are placed into a 30% sucrose sol...

Watch this Scientific Journal Video about Transplantation of Schwann Cells Inside PVDF-TrFE Conduits to Bridge Transected Rat Spinal Cord Stumps to Promote Axon Regeneration Across the Gap at JoVE.com

Transplantation of Schwann Cells Inside PVDF-TrFE Conduits to Bridge Transected Rat Spinal Cord Stumps to Promote Axon Regeneration Across the Gap

This article describes a technique to insert a hollow conduit between the spinal cord stumps after complete transection and fill with Schwann cells (SCs) and injectable basement membrane matrix in order to bridge and promote axon regeneration across the gap.

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord ...

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