I would like to thank: all members of the Lepore, Maragakis and Rothstein labs for helpful discussion; The Paralyzed Veterans of America and the Craig H. Neilsen Foundation for funding.

Methods
1. Cell Preparation
As an example, we will describe the procedure for preparing glial progenitor cells 12 for transplantation because of our experience with this cell type. However, the specifics of the protocol, including medium and use of trypsin for example, will depend on the particular cell type being used for transplantation.
<ol>
	<li>Pre-warm all solutions to 37.0&deg;C in water bath.</li>
	<li>Rinse flask 2X with HBSS. Add 5.0 mL/T-75 flas...

<ol>
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R., Siddique, T., Patterson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+Cu/Zn+superoxide+dismutase+gene+are+associated+with+familial+amyotrophic+lateral+sclerosis.">Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis.</a> Nature. 362, 59-59 (1993).</li><li>Bruijn, L. I., Becher, M. W., Lee, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ALS-linked+SOD1+mutant+G85R+mediates+damage+to+astrocytes+and+promotes+rapidly+progressive+disease+with+SOD1-containing+inclusions.">ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1-containing inclusions.</a> Neuron. 18, 327-327 (1997).</li><li>Gurney, M. E., Pu, H., Chiu, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+neuron+degeneration+in+mice+that+express+a+human+Cu,Zn+superoxide+dismutase+mutation.">Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation.</a> Science. 264, 1772-1775 (1994).</li><li>Howland, D. 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Neurosci. 21, 9246-9246 (2001).</li><li>McDonald, W., Becker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+injury:+promising+interventions+and+realistic+goals.">Spinal cord injury: promising interventions and realistic goals.</a> Am. J. Phys. Med. Rehabi. I82, S38-S38 (2003).</li><li>Sandrow-Feinberg, H. R., Zhukareva, V., Santi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEGylated+interferon-beta+modulates+the+acute+inflammatory+response+and+recovery+when+combined+with+forced+exercise+following+cervical+spinal+contusion+injury.">PEGylated interferon-beta modulates the acute inflammatory response and recovery when combined with forced exercise following cervical spinal contusion injury.</a> Experimental neurology. 223, 439-451 (2010).</li><li>Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+neural+stem+cells.">Mammalian neural stem cells.</a> Science. 287, 1433-1433 (2000).</li><li>Lane, M. A., Lee, K. Z., Fuller, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+circuitry+and+respiratory+recovery+following+spinal+cord+injury.">Spinal circuitry and respiratory recovery following spinal cord injury.</a> Respiratory physiology &#38; neurobiology. 169, 123-123 (2009).</li><li>Lepore, A. C., Rauck, B., Dejea, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+transplantation-based+astrocyte+replacement+is+neuroprotective+in+a+model+of+motor+neuron+disease.">Focal transplantation-based astrocyte replacement is neuroprotective in a model of motor neuron disease.</a> Nature. 11, 1294-1294 (2008).</li><li>Rao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+and+Restricted+Precursors+in+the+Central+Nervous+System.">Multipotent and Restricted Precursors in the Central Nervous System.</a> Anat Rec. 257, 137-137 (1999).</li><li>Rao, M. S., Mayer-Proschel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial-+restricted+precursors+are+derived+from+multipotent+neuroepithelial+stem+cells.">Glial- restricted precursors are derived from multipotent neuroepithelial stem cells.</a> Dev Biol. 188, 48-48 (1997).</li><li>Suzuki, M., Tork, C., Shelley, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+in+disease+onset+and+progression+of+a+rat+model+of+ALS.+Sexual+dimorphism+in+disease+onset+and+progression+of+a+rat+model+of+ALS.">Sexual dimorphism in disease onset and progression of a rat model of ALS. Sexual dimorphism in disease onset and progression of a rat model of ALS.</a> Amyotroph Lateral Scler. 8, 20-20 (2007).</li><li>Lepore, A. C., Haenggeli, C., Gasmi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraparenchymal+spinal+cord+delivery+of+adeno-associated+virus+IGF-1+is+protective+in+the+SOD1G93A+model+of+ALS.">Intraparenchymal spinal cord delivery of adeno-associated virus IGF-1 is protective in the SOD1G93A model of ALS.</a> Brain research. 1185, 256-256 (2007).</li><li>Veldink, J. H., Bar, P. R., Joosten, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+differences+in+onset+of+disease+and+response+to+exercise+in+a+transgenic+model+of+ALS.">Sexual differences in onset of disease and response to exercise in a transgenic model of ALS.</a> Neuromuscul Disord. 13, 737-737 (2003).</li><li>Shumsky, J. S., Lepore, A. 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For studies involving SOD1G93A mice and rats, age- and sex-match the animals within a group, and distribute animals within the same litter to different groups. It is preferable to use all animals from the same sex for both ALS and SCI models because disease processes may differ between males and females; however, it may also be useful to have enough animals from both sexes to detect possible sex-specific effects, as this phenomenon has been reported with SOD1G93A rats 13 and SOD1G93A...

<table border="1">
	<tbody>
		<tr>
			<td>Name of reagent</td>
			<td>Company</td>
			<td>Catalog number</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14170</td>
		</tr>
		<tr>
			<td>0.05% Trypsin</td>
			<td>Gibco</td>
			<td>25300</td>
		</tr>
		<tr>
			<td>Soybean Trypsin Inhibitor (optional)</td>
			<td>Sigma</td>
			<td>T-6522</td>
		</tr>
		<tr>
			<td>Acepromazine maleate (0.7 mg/kg)</td>
			<td>Fermenta Animal Health</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ketamine (95 mg/kg)</td>
			<td>Fort Dodge Animal Health</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine (10 mg/kg)</td>
			<td>Bayer</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>#11 Feather surgical blade</td>
			<td>Electron Microscopy Sciences</td>
			<td>72044-11</td>
		</tr>
		<tr>
			<td>Cotton-tipped applicators (6 inch)</td>
			<td>Fisher</td>
			<td>23-400-101</td>
		</tr>
		<tr>
			<td>Rat-toothed forceps</td>
			<td>Fine Science Tools</td>
			<td>Rat: 11023-15; 
			Mouse: 11042-08</td>
		</tr>
		<tr>
			<td>Medium-sized spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15012-12</td>
		</tr>
		<tr>
			<td>Mini spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-10</td>
		</tr>
		<tr>
			<td>Rongeur</td>
			<td>Fine Science Tools</td>
			<td>Rat: 16121-14; 
			Mouse: 16221-14</td>
		</tr>
		<tr>
			<td>Microknife</td>
			<td>Fine Science Tools</td>
			<td>10056-12</td>
		</tr>
		<tr>
			<td>Needle holders</td>
			<td>Fine Science Tools</td>
			<td>12502-14</td>
		</tr>
		<tr>
			<td>Suture: 4-0</td>
			<td>Vicryl</td>
			<td>S-183</td>
		</tr>
		<tr>
			<td>Staples: 9 mm</td>
			<td>Autoclip</td>
			<td>427631</td>
		</tr>
		<tr>
			<td>Stapler: 9 mm (Reflex #203-1000)</td>
			<td>World Precision Instruments</td>
			<td>5000344</td>
		</tr>
		<tr>
			<td>Warm water pump (T/Pump)</td>
			<td>Gaymar</td>
			<td>P/N 07999-000</td>
		</tr>
		<tr>
			<td>Cyclosporin A: 250.0 mg/5.0 mL ampules</td>
			<td>Novartis/Sandimmune</td>
			<td>NDC 0078-0109-01</td>
		</tr>
		<tr>
			<td>FK-506</td>
			<td>LC Laboratories</td>
			<td>F-4900</td>
		</tr>
		<tr>
			<td>Rapamycin</td>
			<td>LC Laboratories</td>
			<td>R-5000</td>
		</tr>
		<tr>
			<td>Injector</td>
			<td>World Precision Instruments</td>
			<td>UMP2</td>
		</tr>
		<tr>
			<td>Micro 4 Microsyringe Pump Controller</td>
			<td>World Precision Instruments</td>
			<td>UMC4</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instruments</td>
			<td>Kite-R</td>
		</tr>
		<tr>
			<td>10.0 &mu;L Hamilton syringe</td>
			<td>Hamilton</td>
			<td>80030</td>
		</tr>
		<tr>
			<td>Hamilton needles: 33-gauge, 45&deg; bevel, 1 inch</td>
			<td>Hamilton</td>
			<td>7803-05</td>
		</tr>
		<tr>
			<td>Glass 20.0 &mu;L microcapillary pipettes (optional)</td>
			<td>Kimble</td>
			<td>71900-20</td>
		</tr>
	</tbody>
</table>

intraspinal cell transplantation for targeting cervical ventral horn in amyotrophic lateral sclerosis and traumatic spinal cord injury

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1 and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2.
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4. Transgenic mice 4,5 and rats 6 carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R) have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8.
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9.
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A mice and rats, as well as spinal cord injured rats and mice 11.

Watch this Scientific Journal Video about Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury at JoVE.com

Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) ...

intraspinal-cell-transplantation-for-targeting-cervical-ventral-horn

Research

JoVE Journal

Medicine

20.5K visualizações.  Thomas Jefferson University Medical College. Transplante precursoras neurais é uma estratégia promissora para a proteção e / ou substituição perdido / cervical disfuncional neurônios motores frênicos em lesão medular (SCI) e do neurônio motor doença, a esclerose amiotrófica laterais (ALS). Nós fornecemos um protocolo para entrega celular para chifre da medula espinhal cervical ventral em modelos de roedores da ALS e SCI.