We thank the Rat Resource and Research Center, University of Missouri, Columbia, Missouri, for providing GFP rats; Neuralstem Inc. for providing human neural stem cells. This work was supported by the Veterans Administration, NIH (NS09881), Canadian Spinal Research Organization, The Craig H. Neilsen Foundation, and the Bernard and Anne Spitzer Charitable Trust.

All animal protocols are approved by VA-San Diego Institutional Animal Care and Use Committee (IACUC). NIH guidelines for laboratory animal care and safety are strictly followed. Animals have free access to food and water throughout the study and are adequately treated for minimizing pain and discomfort.
1. Preparation of Fibrin Components Containing Growth Factor Cocktails
<ol>
	<li>Dissolve 25 mg rat fibrinogen in 0.5 ml PBS to obtain 50 mg/ml 2x stock solution (see Materials table). Fibrinogen is difficult to dissolve and should be placed into a 37 &#176;C incubator or waterbath and shaken every 5-10 min for 1-2 hr.</li>
	<li>Dissolve 100 UN rat thrombin in 2 ml of 10 mM sterile CaCl2 to obtain 50 U/ml 2x stock solution.</li>
	<li>Dissolve growth factors in PBS at the following concentrations to obtain 100 &#956;l stock solution: 1 mg/ml: BDNF; NT-3 (high stock concentration as intrathecal infusion in vivo19); 200 &#956;g/ml: GDNF; IGF; bFGF; EGF; PDGF; aFGF; HGF (about 1,000x higher than used for cell culture).</li>
	<li>Dissolve 25 mg MDL28170 (calpain inhibitor) in 1.3 ml DMSO to obtain 50 mM DMSO stock solution, and then dilute 50x in PBS to obtain 1 mM stock solution.</li>
	<li>Mix 100 &#956;l of each growth factor component and 100 &#956;l of MDL28170 (1 mM stock solution) to produce 1,000 &#956;l growth factor cocktail solution (Figure 1).</li>
	<li>Mix 500 &#956;l fibrinogen 2x stock solution with 500 &#956;l growth factor cocktail to obtain 25 mg/ml fibrinogen working solution containing growth factors and aliquot into 10 or 20 &#956;l for storage at -70 &#176;C. The solution is stable for up to 12 months at -70 &#176;C.</li>
	<li>Similarly, mix 500 &#956;l thrombin 2x stock solution with 500 &#956;l growth factor cocktail to obtain 25 U/ml thrombin working solution containing growth factors and aliquot into similar 10 or 20 &#956;l volumes for storage at -70 &#176;C for up to 12 months.</li>
	<li>On the day of grafting, place fibrinogen- and thrombin-containing growth factor cocktails on ice until mixed with cells.</li>
</ol>
2. T3 Spinal Cord Transection
<ol>
	<li>Anesthetize adult female Fischer 344 rats or athymic nude rats using acceptable methods. Subjects are deeply anesthetized, when responsiveness to tail and paw pinch are completely absent. Note: We typically use 150-200 g Fischer rats or 180-220 g athymic rats, and an anesthesia cocktail (2 ml/kg) of ketamine (25 mg/ml), xylazine (1.3 mg/ml) and acepromazine (0.25 mg/ml).</li>
	<li>Apply eye ointment to prevent dryness or injury of eyes while the animals are under anesthesia.</li>
	<li>Shave the upper thoracic area and clean the skin with Betadine.</li>
	<li>Cut the skin and muscle using #15 blade, expose T3 vertebra using a surgical retractor, and perform a laminectomy at T3 vertebra to expose T3/4 spinal cord using a rongeur.</li>
	<li>Make a longitudinal midline incision in the dura of approximately 2 mm length using a #11 blade.</li>
	<li>Place iridectomy scissors underneath the dura to cut the right lateral entire spinal cord. Make another cut on the same side 1.5 mm more cadually.</li>
	<li>Aspirate spinal cord tissue between the two cut segments with a blunt 23 G&#160;needle connected to moderate intensity vacuum. Use visual verification to ensure complete transection ventrally and laterally. This will complete a lateral hemisection.</li>
	<li>To extend the lesion to a full-width spinal cord transection, place mirror incisions into the left hemicord. Attempt to leave the dura intact, other than the first cut, to retain the firm graft/fibrin matrix in the lesion site when grafted two weeks later.</li>
	<li>Suture the muscle, apply antibiotic powder and staple the skin.</li>
	<li>Inject lactated Ringer&#39;s (3 ml), Bannamine (2.5-5 mg/kg) and Ampicillin (80-100 mg/kg) (see Materials table) immediately after surgery and maintain rats in a warm incubator until thermoregulation is re-established.</li>
	<li>Empty the bladder and inject the Ringer&#39;s and Ampicillin solution twice a day for about two weeks until the establishment of reflex bladder emptying (Figure 1B).</li>
</ol>
3. Preparation of Freshly Dissociated Embryonic Day 14 Spinal Cord Neural Stem Cells
<ol>
	<li>Obtain inbreed transgenic GFP F344 rats (F344-Tg(UBC-EGFP)F455Rrrc) from Rat Resource and Research Center, University of Missouri.</li>
	<li>Inject 40 &#956;g des-Gly10, [D-Ala6]-Leuteinizing Hormone Releasing Hormone Ethylamide (LHRH) diluted in PBS at a concentration of 200 &#956;g/ml intraperitoneally (IP) per mature female rat (8 weeks or older) after 2-3 hr light induction for synchronization of embryo collection.</li>
	<li>Mate with a mature GFP homozygous or heterozygous male rat overnight 4 days after injection.</li>
	<li>Collect embryos at days 13.5-14.5 postcoitus (p.c.) in cold HBSS buffer, examine GFP expression under a &#34;NightSea&#34; flashlight and filter glasses (BLS1 - BlueStar Flashlight and Model VG1 barrier filter) or a fluorescent microscope.</li>
	<li>Dissect spinal cord out aseptically from each GFP-positive fetus and remove overlying meninges and attached spinal ganglia with iridectomy scissors and fine jewelers forceps.</li>
	<li>Collect the dissected spinal cords into 2 ml cold HBSS buffer in a 15 ml cornical tube and keep on ice.</li>
	<li>Follow reference #20 to dissociate dissected spinal cords:
	<ol>
		<li>Briefly, add 2 ml of 0.25% Trypsin-EDTA to the tube containing dissected spinal cords (several cords can be digested together, 1 cord can generate enough cells for graft of one subject) and digest at 37 &#176;C for 10-12 min.</li>
		<li>Stop the trypsin reaction by adding 10 ml of DMEM containing 10% FBS to the tube and centrifuge the tissue at 2,500 rpm for 2 min.</li>
		<li>Resuspend tissue in 2 ml NeuroBasal medium containing 2% B27, and triturate the spinal cord tissue using progressively smaller fire-polished Pasteur pipets.</li>
		<li>Centrifuge the cells at 2,500 rpm for 2 min, resuspend in 2 ml of NeuroBasal medium containing B27, and filter cells with a 40 &#956;m cell filter strainer.</li>
	</ol>
	</li>
	<li>Count the cells with a hemocytometer and divide the cells equally into two Eppendorf tubes.</li>
	<li>Centrifuge the cells and completely remove supernatant (1-2 min are required to withdraw all supernatant with a low vacuum tip) so the growth factor cocktails will not be diluted by remaining supernatant after cell re-suspension.</li>
	<li>Resuspend each half of the cells in the fibrinogen or thrombin working solution containing growth factors, respectively, at a concentration of 250,000 cells/&#956;l (cell pellet counts for about &#8531; final volume) and place them on ice prior transplantation (Figure 1A). Note that fibrinogen may spontaneously gel when mixed with cells before combining with thrombin at the lesion/graft site; to avoid premature gellation, mix cells in fibrinogen immediately before cell grafting in vivo.</li>
</ol>
4. Transplantation
<ol>
	<li>Reexpose the lesioned T3/4 spinal cord two weeks after spinal cord transection.</li>
	<li>5 &#956;l fibrinogen-cells and 5 &#956;l thrombin-cells could be directly injected into nine points of the lesion site through sealed scar tissue and intact dura without re-opening the lesion site.</li>
	<li>Use a 27 G needle to create 9 holes on the surface of the scar tissue and intact dura 1-2 weeks post lesion: 3 in center (one in middle point and two in laterals, and spaced 0.5 mm apart) and 3 in both rostral and 3 in caudal interface (about 0.5 mm rostral or caudal to the center of the lesion).</li>
	<li>Then inject half volume of fibrinogen cell solution into three central points of the lesion site and a quarter volume into 3 points of the rostral interface and a quarter volume into 3 points of the caudal interface.</li>
	<li>Then, immediately inject throbmbin cells into the same spots. The mixture of fibrinogen cells and thrombin cells inside the lesion site will form fibrin gel to hold cells into lesion site and the growth factor cocktails will support the survival of cells. Cells are injected using pulled glass pipettes connected to a PicoSpritzer (General Valve, Inc.).</li>
	<li>Transplant cultured human NSCs21 in the same procedure described above, and use fibrin and growth factor cocktails made from human reagents.</li>
</ol>

<ol>
	<li>Ramon y Cajal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Degeneration+and+regeneration+of+the+nerve+system.">In Degeneration and regeneration of the nerve system.</a> , (1991).</li><li>Jakeman, L. B., Reier, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+projections+between+fetal+spinal+cord+transplants+and+the+adult+rat+spinal+cord:+a+neuroanatomical+tracing+study+of+local+interactions.">Axonal projections between fetal spinal cord transplants and the adult rat spinal cord: a neuroanatomical tracing study of local interactions.</a> J Comp Neurol. 307, 311-334 (1991).</li><li>Bamber, N. I., Li, H., Aebischer, P., Xu, X. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+spinal+cord+tissue+in+mini-guidance+channels+promotes+longitudinal+axonal+growth+after+grafting+into+hemisected+adult+rat+spinal+cords.">Fetal spinal cord tissue in mini-guidance channels promotes longitudinal axonal growth after grafting into hemisected adult rat spinal cords.</a> Neural Plast. 6, 103-121 (1999).</li><li>Theele, D. P., Schrimsher, G. W., Reier, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+growth+and+fate+of+fetal+spinal+iso-+and+allografts+in+the+adult+rat+injured+spinal+cord.">Comparison of the growth and fate of fetal spinal iso- and allografts in the adult rat injured spinal cord.</a> Exp Neurol. 142, 128-143 (1996).</li><li>Giovanini, M. A., Reier, P. J., Eskin, T. A., Wirth, E., Anderson, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+human+fetal+spinal+cord+grafts+in+the+adult+rat+spinal+cord:+influences+of+lesion+and+grafting+conditions.">Characteristics of human fetal spinal cord grafts in the adult rat spinal cord: influences of lesion and grafting conditions.</a> Exp Neurol. 148, 523-543 (1997).</li><li>Wictorin, K., Bj&#246;rklund, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+outgrowth+from+grafts+of+human+embryonic+spinal+cord+in+the+lesioned+adult+rat+spinal+cord.">Axon outgrowth from grafts of human embryonic spinal cord in the lesioned adult rat spinal cord.</a> Neuroreport. 3, 1045-1048 (1992).</li><li>Vroemen, M., Aigner, L., Winkler, J., Weidner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neural+progenitor+cell+grafts+survive+after+acute+spinal+cord+injury+and+integrate+along+axonal+pathways.">Adult neural progenitor cell grafts survive after acute spinal cord injury and integrate along axonal pathways.</a> Eur J Neurosci. 18, 743-751 (2003).</li><li>Blits, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vector-mediated+transduction+of+neural+progenitor+cells+before+implantation+into+injured+spinal+cord+and+brain+to+detect+their+migration,+deliver+neurotrophic+factors+and+repair+tissue.">Lentiviral vector-mediated transduction of neural progenitor cells before implantation into injured spinal cord and brain to detect their migration, deliver neurotrophic factors and repair tissue.</a> Restor Neurol Neurosci. 23, 313-324 (2005).</li><li>Lepore, A. 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We have nothing to disclose.

One of the major hurdles for NSC transplantation in the injured spinal cord is poor survival in the lesion center. Any gaps or cavities in the lesion site could potentially reduce or weaken the formation of functional neuronal relays between supraspinal axons and separated spinal cord segments below the injury. In addition, poor survival of grafted NSCs may affect their integration with host tissue, and therefore reduce connectivity of grafted neurons with host neurons. Potential mechanisms to explain cell loss include t...

Spinal cord injury (SCI) often damages not only white matter tracts that carry signals to and from the brain, but also the central gray matter, causing segmental loss of interneurons and motor neurons. The consequence of SCI is loss of both motor and sensory function below the lesion. Unfortunately, the adult central nervous system (CNS) does not spontaneously regenerate, resulting in permanent functional deficits1. Therefore, reconstruction of the injured adult spinal cord and improvement in motor, sensory and autonomic function is an important goal of SCI research. Neural stem cells (NSCs), whether directly isolated from embryonic or adult CNS, are compelling candidate cells to replace lost neurons and glia. Moreover, these cells have the potential to form new functional relays to restore axonal conduction across a lesion site2,3.
To date, there has not been a detailed elucidation of the anatomical, electrophysiological and behavioral effects of neuronal relay formation by transplanted NSCs after severe SCI. There are several reasons for this: first, transplanted NSCs or fetal CNS tissue survive poorly when grafted into large lesion cavities. Previous studies show substantial early cell loss, leaving large empty cystic lesion cavities4,5. In some studies the grafted cells would subsequently divide and fill the lesion cavity4,5, but this might occur after a delay of days to weeks, and subsequent lesion site filling might not be complete or consistent. Second, an efficient tracking system that provides sound data on cellular survival, differentiation/maturation, and outgrowth of transplanted NSCs was lacking. Most early studies utilized antegrade and retrograde labeling to trace axonal projections from transplants2,3. However, these techniques only partially and often unclearly labeled axonal projections arising from grafted cells, and tracer methods are subject to artifacts caused by dye leakage beyond the implanted cells. Other groups used human specific neuronal markers to label axonal projections after transplantation of human fetal NSCs into injured rodent spinal cord5,6. However, in those studies, the xenografts did not consistently survive well. Recently, viral &#160;delivery of the GFP reporter gene was used to label cultured NSCs7,8. However, GFP expression was often inconsistent and can be down-regulated7. Recently, the use of transgenic donor mice or rats stably expressing the reporter gene, GFP, or the human placental alkaline phosphatase, has dramatically improved the tracking of transplanted neural stem cells/progenitors in vivo9,11. Third, several studies indicate that in vitro cultured rat NSCs derived from either embryonic or adult CNS exclusively differentiate into glial lineages when transplanted into the milieu of the intact or injured adult spinal cord7,12,13, despite the fact that these neural stem cells are capable of differentiating into both neurons and glia in vitro, indicating that local environments may dictate the fate of stem cells. Alternatively, cultured NSCs, especially those derived from adult CNS, may have intrinsic defaulty property to differentiate into glial lineages in vivo13.
Because of the limitations discussed above, our group recently developed a new protocol to improve embryonic NSC tracking, survival, and differentiation/maturation in the severely injured adult spinal cord. Briefly, we began with a stable transgenic Fischer 344 rat inbreed line expressing a GFP reporter gene that sustains GFP expression after in vivo transplantation14. Next, we used freshly isolated NSCs from embryonic day 14 Fischer 344 spinal cord, a stage of development that retains the potential to generate both neurons and glia. Finally, we embedded freshly dissociated NSCs into a fibrin matrix containing growth factors15-17 to retain the cells and evenly distribute them within a large lesion cavity, aiming to support graft cell survival, differentiation and integration. Grafts were placed into sites of T3 complete transection, two weeks after spinal cord injury. These grafted cells consistently filled complete transection sites and differentiated into abundant neurons that extended large numbers of axons into host spinal cord over long distances18. Similar results were obtained using cultured human neural stem cell grafts to immuno-deficient rats18.

<table border="1">
 <tbody>
 <tr>
 <td>Reagents</td>
 <td>Company</td>
 <td>Catalogue</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>Fibrinogen(rat)</td>
 <td>Sigma</td>
 <td>F6755-25MG</td>
 <td>2 hr at 37 &deg;C to dissovle 
 Stock Concentration: 50 mg/ml 
 Final Concentration: 25 mg/ml</td>
 </tr>
 <tr>
 <td>Thrombin (rat)</td>
 <td>Sigma</td>
 <td>T5772-100UN</td>
 <td>Dissovle in 10 mM CaCl2 
 Stock Concentration: 50 U/ml 
 Final Concentration: 25 mg/ml</td>
 </tr>
 <tr>
 <td>bFGF (human)</td>
 <td>Sigma</td>
 <td>F0291 (25 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>EGF (murine)</td>
 <td>Sigma</td>
 <td>E1257 (0.1 mg)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>BDNF(human)</td>
 <td>Peprotech</td>
 <td>452-02 (1 mg)</td>
 <td>Stock Concentration: 1000 ng/&mu;l 
 Final Concentration: 50 ng/&mu;l</td>
 </tr>
 <tr>
 <td>NT-3 (human)</td>
 <td>Peprotech</td>
 <td>452-03 (1 mg)</td>
 <td>Stock Concentration: 1000 ng/&mu;l 
 Final Concentration: 50 ng/&mu;l</td>
 </tr>
 <tr>
 <td>GDNF (rat)</td>
 <td>Sigma</td>
 <td>G1401 (10 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>IGF-1(mouse)</td>
 <td>Sigma</td>
 <td>I8779 (50 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>aFGF (human)</td>
 <td>Sigma</td>
 <td>F5542 (25 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l </td>
 </tr>
 <tr>
 <td>PDGF-AA (human)</td>
 <td>Sigma</td>
 <td>P3076 (10 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>HGF (human)</td>
 <td>Sigma</td>
 <td>H9661 (5 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>MDL28170</td>
 <td>Sigma</td>
 <td>M6690 (25 mg)</td>
 <td>Stock in DMSO 
 Stock Concentration: 1 mM 
 Final Concentration: 50 &mu;M</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Millipore</td>
 <td>BSS-1005-B</td>
 <td>Stock Concentration: 1X 
 Final Concentration: 1X</td>
 </tr>
 <tr>
 <td>DMSO</td>
 <td>Sigma</td>
 <td>D2650</td>
 <td></td>
 </tr>
 <tr>
 <td>Ketamine</td>
 <td>Putney</td>
 <td>26637-411-01</td>
 <td>40-80 mg/kg 
 Stock Concentration: 100 mg/ml 
 Final Concentration: 25 mg/ml</td>
 </tr>
 <tr>
 <td>Xylazine</td>
 <td>Lloyd</td>
 <td>0410,</td>
 <td>2.5-8 mg/ml 
 Stock Concentration: 100 mg/ml 
 Final Concentration: 1.3 mg/ml</td>
 </tr>
 <tr>
 <td>Acepromazine</td>
 <td>Butler</td>
 <td>003845</td>
 <td>0.5-4 mg/ml 
 Stock Concentration: 10 mg/ml 
 Final Concentration: 0.25 mg/ml</td>
 </tr>
 <tr>
 <td>Betadine</td>
 <td>Healthpets</td>
 <td>BET16OZ</td>
 <td></td>
 </tr>
 <tr>
 <td>Ringers</td>
 <td>Abbott</td>
 <td>04860-04-10</td>
 <td>2-3 ml/inj</td>
 </tr>
 <tr>
 <td>Banamine</td>
 <td>Schering-Plough</td>
 <td>0061-0851-03</td>
 <td>2.5 -5 mg/kg 
 Stock Concentration: 50 mg/ml 
 Final Concentration: 0.5 mg/ml</td>
 </tr>
 <tr>
 <td>Ampicillin</td>
 <td>Sandoz</td>
 <td>0781-3404-85</td>
 <td>80-100 mg/kg 
 Final Concentration: 50 mg/ml</td>
 </tr>
 <tr>
 <td>LH-RH</td>
 <td>Sigma</td>
 <td>L4513</td>
 <td>200 &mu;g/kg 
 Final Concentration: 200 &mu;g/ml</td>
 </tr>
 <tr>
 <td>HBSS</td>
 <td>Gibco</td>
 <td>14175-096</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>Gibco</td>
 <td>25200056 </td>
 <td>Stock Concentration: 0.25 % 
 Final Concentration: 0.125 %</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Gibco</td>
 <td>11995073 </td>
 <td></td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Gibco</td>
 <td>16000044 </td>
 <td>Stock Concentration: 100 % 
 Final Concentration: 10 %</td>
 </tr>
 <tr>
 <td>Neurobasal Medium</td>
 <td>Gibco</td>
 <td>21103049 </td>
 <td></td>
 </tr>
 <tr>
 <td>B27</td>
 <td>Gibco</td>
 <td>17504044</td>
 <td>Stock Concentration: 100x 
 Final Concentration: 1x</td>
 </tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Note, use human reagents for grafts of human NSCs</td>
 </tbody>
</table>

promotion of survival and differentiation of neural stem cells with fibrin and growth factor cocktails after severe spinal cord injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

GFP immunohistological labeling demonstrates that grafted rat NSCs resuspended in phosphate buffered saline (PBS) (lacking a fibrin matrix and growth factors) survived poorly in the T3 transection site, only attaching to the lesion/host margin and leaving most of the lesion site empty without grafted cells (Figure 2A). The survival of NSCs improved when co-grafted with fibrin matrices alone, but the graft did not completely fill up a large lesion cavity (data not shown). Therefore, we embedded NSCs into ...

Watch this Scientific Journal Video about Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury at JoVE.com

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

promotion-survival-differentiation-neural-stem-cells-with-fibrin

Research

JoVE Journal

Neuroscience

12.3K 조회수.  Veterans Administration Medical Center, San Diego.  성장 인자를 함유하는 피브린 매트릭스는 완전한 척수 절개 부위로 그라프 신경 줄기 세포를 유지하기 위해 사용되었다. 이식 세포가 완전히 병변 캐비티를 채우고 장거리 호스트 척수로 축삭을 확장 신경 세포를 포함한 여러 신경 세포 유형으로 분화. 

비디오: Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...