Name: Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury (Video) | JoVE Journal
Uploaded: 2014-12-17T13:00:00.000+00:00
Duration: 10 min 56 s
Description: Learn neural stem cell transplantation in a mouse spinal cord injury contusion model using PKH 26-labeled precursor cells. This method enables tracking of engrafted cells and studying regeneration potential.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The Authors acknowledge the economic support by FAIP (Federazione Associazioni Italiane Paraplegici), &#8220;Neurogel-en-Marche&#8221; Foundation (France), Fondazione &#8220;La Colonna&#8221;.

NOTE: All the procedures were approved by the Review Committee of the University of Milan and met the Italian Guidelines for Laboratory Animals in compliance with European Communities Directive dated November 1986 (86/609/EEC).
1. Preparation of Cells for Transplantation
NOTE : Use neural stem cells between the 5th and the 9th passage in culture for these experiments; test the cultures for proliferation and differentiation ability before being labeled for transplantation. Determine the extent of differentiation by immunocytochemistry 12.
<ol>
	<li>Resuspend cells to a concentration of 1 x 106 cells/150 &#956;l (transplant 1 x 106 cells per mouse). Prepare at least 1.2 x 106 cells per mouse because an excess of cells is required for syringe loading purposes.</li>
	<li>Wash cells 3 times using neural stem cell medium 13 in a 10 ml conical vial. In each washing step, precipitate cells by centrifugation (500 x g for 5 min, RT).</li>
	<li>Count the cells before the final spin.</li>
	<li>Centrifuge the cells (500 x g for 5 min), and then aspirate the supernatant, being careful not to remove any cells but leaving no more than 25 &#956;l of supernatant.</li>
	<li>Prepare a 2x cell suspension by adding 1 ml of Diluent C to the cell pellet and resuspend with gentle pipetting.</li>
	<li>Immediately before staining, prepare a 2x Dye Solution (4 x 10&#8211;6 M) in Diluent C by adding 4 &#181;l of the PKH26 ethanol dye solution to 1 ml of Diluent C in a tube and mix well to disperse.</li>
	<li>Rapidly add the 1 ml of 2x cell suspension to 1 ml of 2x Dye Solution and immediately mix the sample by pipetting (final cell density will be 1.2 x 107 cells/ml and 2 x 10&#8211;6 M PKH26).</li>
	<li>Incubate the cell/dye suspension for 1-5 min.</li>
	<li>Stop the staining by adding an equal volume (2 ml) of 1% BSA solution in HBSS and incubate for 1 min.</li>
	<li>Centrifuge cells (500 x g for 10 min) and carefully remove the supernatant.</li>
	<li>Resuspend cell pellet in 10 ml of HBSS and centrifuge (500 x g for 5 min).</li>
	<li>Wash the cell pellet 2 more times with 10 ml of complete medium to ensure removal of unbound dye.</li>
	<li>Resuspend the cell pellet in 10 ml of complete medium for assessment of cell recovery, cell viability and fluorescence intensity. If cells are needed for transplantation, wash and resuspend them in a sterile physiological solution at a concentration of 3.3 x 105 cells/50 &#956;l.</li>
</ol>
2. Preparation for Surgery
<ol>
	<li>Clean and sterilize surgery equipment.</li>
	<li>Prepare the surgery area by wiping with an aseptic agent. Set up the IH Impactor device.</li>
	<li>Prepare the anesthesia equipment: the Active Gas Scavenger with VetScav Filter Weighing Device, continuous flow Induction Chamber, Oxygen Generator, Low Flow O2 Meter for Mice. Clean the induction chamber and the mask used during surgery.</li>
	<li>Anesthetize animals with 2.5% (v/v) isoflurane in oxygen (1 L/min), and wait 5 min after the flow induction for the drug to take effect. Check if the whiskers are twitching or if there is slow hind limb withdrawal in response to pinching the footpad. During the surgery, reduce the isoflurane concentration to 2.0% (v/v) isoflurane in oxygen (1 L/min).</li>
</ol>
3. Preparation of Mice for Surgery and Transplantation
<ol>
	<li>Keep the male adult CD1 mice (25&#8211;30 g) for at least 3 days before the experiments in standard conditions (22 &#177; 2 &#176;C, 65% humidity, and artificial light between 08:00 a.m. to 08:00 p.m.). 
	NOTE: The entire procedure from surgical preparation to suturing will take about 40 min.
	<ol>
		<li>To identify the animal, mark the tail by means of water resistant colored ink.</li>
	</ol>
	</li>
	<li>Use an electric clipper to cut the dorsal hair of the mouse from the neck, about at T2 level, to the lumbar area.</li>
	<li>Sterilize the prepared area with an iodide solution and ethanol (70% in sterile water).</li>
	<li>Treat the animal with a sub cutaneous (s.c.) injection of 200 &#956;l gentamycin (1 mg/ml in sterile saline solution).</li>
	<li>Apply ophthalmic lubricant to both animal eyes to prevent desiccation and inject buprenorphine (subcutaneously, 0.03 mg/kg) to alleviate pain.</li>
</ol>
4. Laminectomy
<ol>
	<li>Place the mouse on a slide warmer to avoid the problem of hypothermia during the surgery. Position the animal with the dorsal side up.</li>
	<li>Make a vertical incision with a scalpel over the region of interest, from T7 to T12.</li>
	<li>Hold the skin and superficial fat pad by using standard forceps (usually found in the space between the 5th and 6th thoracic dorsal processes).</li>
	<li>Count the process under the vessel as T6 then move to T7.</li>
	<li>Place a little bearing under the ventral side of the mouse to increase the curvature of the spine. Immobilize the spinal cord by blocking it with the Graefe forceps.</li>
	<li>Cut the paravertebral muscles bilaterally from T7 and T10 vertebral level by using the scalpel until the dorsal surface of the lamina contacts the scalpel tip.</li>
	<li>Use the scalpel to tick off the junction from T7 until T10. Stop in the space between T8 and T9 spiny protrusions. Cut the tissues between T8-T9 and T9-T10 with the micro scissors.
	<ol>
		<li>Use the Rongeur to remove the T9 process. Expose the junction by carefully scraping away the muscle layer with the micro scissors. Continue until bone is exposed.</li>
	</ol>
	</li>
	<li>Use the forceps to remove muscles from the lamina and open a small space between the vertebrae. Gently insert the micro scissors under the bone, cut the lamina on both sides and remove this part with forceps.</li>
	<li>Remove the lamina with the forceps to expose the cord. Be sure not to leave any free or jagged bone fragments behind; remove them with the Rongeur.</li>
	<li>Use the small tip forceps to remove the periosteum as well as any bone fragments or muscles that may be near the incision.</li>
	<li>Remove the top half of the T9 dorsal process and proceed to the IH Impactor device protocol.</li>
</ol>
5. IH Impactor Device Protocol (Contusion)
<ol>
	<li>Place the mouse in the middle of the stabilization platform of the IH Impactor device.</li>
	<li>Block the animal with the two teeth forceps connected with the stabilization platform by two joint positioning arms (left arm for the thoracic vertebra, right arm for cervical vertebra).</li>
	<li>Use the rostral arm to grasp the lateral edge of the rostral vertebral body (T8).</li>
	<li>Manipulate the caudal arm in the same manner to grasp the T10 vertebral body.</li>
	<li>Place the stabilization platform in the device and lower the tip (diameter 0.75 mm) as close to the cord as possible without touching it.</li>
	<li>Lift the tip three complete turns before initiating the impact.</li>
	<li>Perform the contusion with the impactor set to deliver a force of 60 kdyn at 100 mm/sec.</li>
</ol>
6. Sutures and Post-care
<ol>
	<li>Suture the incision with a 4/0 absorbable suture thread. Cover the exposed spinal cord at the site of removed lamina; suture the tissue at the extremities of the cut by means of a small needle. Put the two stitches immediately above and under the site of spinal cord exposure.</li>
	<li>Close the skin using two or three Reflex clips without pinching off the underlying muscles.</li>
	<li>Hydrate the mouse post-surgery with 2 ml of saline solution subcutaneously injected in the lower back.</li>
	<li>Place the mouse back in a pre-warmed cage to avoid hypothermia during surgical recovery. Place cages on heating pads.</li>
	<li>Monitor the mice during the acute phase post-injury by checking the size bladder, the suture and the animal weight twice a day.&#160;Gently squeeze bladder (twice a day for 7 days) to avoid urinary tract&#8217;s infections (urine could be cloudy, bloody or containing any precipitates).</li>
	<li>Treat mice once a day with a saline solution injection (2 ml for two days post-surgery s.c. injection) and antibiotic (gentamycin 0.2 ml; s.c. injection) for five days post injury.</li>
	<li>Treat mice for post-operative analgesia with buprenorphine (0.1 mg/kg; twice a day) for 3 days post injury.</li>
</ol>
7. Tail Vein Injection of Cells
NOTE: In the following step the procedure for injecting the cells into the tail vein is demonstrated. Cells could be also administered with an intraspinal transplant by using a stereotaxic frame 15-16, or into the cisterna magna 17. It is important to consider that other cell types could be transplanted with this method, such as mesenchymal stromal cells (e.g., bone marrow mesenchymal stem cells, adipose derived stem cells, amniotic fluid cells). Furthermore, other treatment options such as nanoparticles can be injected via the tail vein after the spinal cord injury.
<ol>
	<li>Use 70% ethanol and PBS to clean the needle before use. Handle the needle and the syringe only with sterile gloves.</li>
	<li>Resuspend the cells in the test tube and load 75 &#956;l of cells into a 0.3 ml syringe (29 G&#160;needle and 0.33 cc syringe).</li>
	<li>Make sure that no bubbles are present within the cell suspension. Keep the syringe in a horizontal position to avoid cell sedimentation.</li>
	<li>Place the mouse underneath the heat lamp to dilate the tail veins.</li>
	<li>Grab the mouse and gently pull it into the mouse restrainer to visualize the lateral tail vein as a narrow blue line.</li>
	<li>Clean the tail with an alcohol swab. Once the vein is visualized, grab the tail vein between the middle finger and thumb of the left hand.</li>
	<li>Bring the needle to the surface at a 15&#176; angle from the horizon and make sure the bevel is up.</li>
	<li>Inject 50 &#956;l of cells at a rate of .33 &#956;l/sec. After the injection, delay the retraction of the needle by 10 sec. Retract the needle slowly and pay attention to the possible efflux of cell suspension.</li>
	<li>Clean the syringe as in step 7.1 between loads.</li>
</ol>
8. Behavioral Tests and Hind Limb Function
<ol>
	<li>Place the mouse in the open field.</li>
	<li>Evaluate locomotor function and hind limb recovery after contusion with the open field test according to the Basso mouse scale (BMS) 18 . 
	NOTE: Neurological function must be evaluated periodically, from day 3 after injury for 4 weeks 18.</li>
</ol>
9. Perfusion
<ol>
	<li>At the end of the experimental period, anesthetize animals by an intraperitoneal injection of sodium pentobarbital (65 mg/kg). Use toe pinches to evaluate the anesthesia level and proceed only after mouse is unresponsive to the noxious stimuli.</li>
	<li>Restrain the animal in a supine position on a surgery plane.</li>
	<li>Cut through the integument and abdominal wall just beneath the rib cage. Separate the diaphragm from the liver.</li>
	<li>Use the scissors to cut the diaphragm to expose the pleural cavity.</li>
	<li>Cut the sides of the rib cage until the collar bones.</li>
	<li>Use the hemostats to clamp the xiphoid cartilage and place the hemostat over the head.</li>
	<li>Hold the bottom third of the heart on a transversal plane with the forceps. Insert the needle into the left ventricle.</li>
	<li>Use a hemostat to clamp the heart. This secures the needle and prevents leakage.</li>
	<li>Use the scissors to cut the right atrium.</li>
	<li>Allow the PBS to pump (18 ml/min) through the animal. Maintain this pressure throughout the buffer infusion period (3-4 min; corresponding to about 70 ml PBS). Continue until the heart is clean.</li>
	<li>Switch the stopcock to allow the fixative (4% Paraformaldehyde in distilled water, 4%PFA) through the pump. Allow the 4% PFA to pump through the animal for approximately 8-10 min (300 ml). Gradually increase the pressure to reach a maximum of 30 ml/min. A hardened liver is the best indication of a successful perfusion.</li>
</ol>
10. Tissue Collection and Processing, Histology and Iimmunohistochemistry
<ol>
	<li>Dissect spinal cords from T5 to L1. Then post-fix the tissue in 6 ml in 4% PFA (O/N at 4 &#176;C).</li>
	<li>Put the same tissue in a solution with 30% sucrose for 72 hr at 4 &#176;C in order to cryo protect it and to prevent the formation of crystals during freezing.</li>
	<li>Quick freeze the cord using dry ice and store it at -80 &#176;C.</li>
	<li>Section by means of a cryostat with 15 &#181;m thickness and collect sections onto glass slides and continue for immunocytochemistry.</li>
	<li>Rinse sections with 200 &#956;l of PBS for each slide (3 times; 5 min each, RT).</li>
	<li>Permeabilize each slide with 200 &#956;l of 10% NGS and 0.2% Triton X-100 in PBS for 1 hr at RT.</li>
	<li>Rinse sections with 200 &#956;l of PBS for each slide (3 times; 5 min each, RT).</li>
	<li>Block non-specific sites with 200 &#956;l of blocking solution for slide (5% NGS; 0.1% Triton X-100 in PBS) for 30 min (RT).</li>
	<li>Incubate each slide with 200 &#956;l of primary antibodies O/N at 4 &#176;C (dilute antibody in 5% NGS; 0.1% Triton X-100 in PBS).</li>
	<li>Wash sections with 200 &#956;l of PBS for each slide (3 times; 5 min&#160;each; RT).</li>
	<li>Incubate with appropriate secondary antibodies for 2 hr at RT.</li>
	<li>Wash sections with 200 &#956;l of PBS for each slide (3 times; 5 min&#160;each; RT).</li>
	<li>Stain nuclei with 200 &#956;l of 4&#39;,6-diamidin-2-phenilindole (DAPI) (1 &#181;g/ml final concentration, 10 min at RT).</li>
	<li>Mount by using the FluorSave Reagent and analyse by confocal microscopy. 
	NOTE: In control determinations, primary antibodies must be omitted and replaced with equivalent concentrations of unrelated IgG of the same subclass. Microtubule-Associated Protein 2 primary antibody was used.</li>
</ol>

<ol><li>Cord Injury Statistical Center: spinal cord injury facts and figures at glance. , 
 
 <a target="_blank" href="https://www.nscisc.uab.edu/PublicDocuments/fact_figures_docs/Facts%202013.pdf">https://www.nscisc.uab.edu/PublicDocuments/fact_figures_docs/Facts%202013.pdf</a> (2013).</li>
<li>Yip, P. K., Malaspina, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spinal+cord+trauma+and+the+molecular+point+of+no+return%5BTitle%5D)+AND+%22Molecular+Neurodegeneration%22%5BJournal%5D)">Spinal cord trauma and the molecular point of no return.</a> Molecular Neurodegeneration. 7, 6(2012).</li>
<li>Fehlings, M. G., Cadotte, D. W., Fehlings, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+series+of+systematic+reviews+on+the+treatment+of+acute+spinal+cord+injury%3A+a+foundation+for+best+medical+practice%5BTitle%5D)+AND+%22J+Neurotrauma%22%5BJournal%5D)">A series of systematic reviews on the treatment of acute spinal cord injury: a foundation for best medical practice.</a> J Neurotrauma. 28 (8), 1329-1333 (2011).</li>
<li>Furlan, J. C., Noonan, V., Cadotte, D. W., Fehlings, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Timing+of+decompressive+surgery+of+spinal+cord+after+traumatic+spinal+cord+injury%3A+an+evidence-based+examination+of+pre-clinical+and+clinical+studies%5BTitle%5D)+AND+%22J+Neurotrauma%22%5BJournal%5D)">Timing of decompressive surgery of spinal cord after traumatic spinal cord injury: an evidence-based examination of pre-clinical and clinical studies.</a> J Neurotrauma. 28 (8), 1371-1399 (2011).</li>
<li>Sekhon, L. H., Fehlings, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidemiology%2C+demographics%2C+and+pathophysiology+of+acute+spinal+cord+injury%5BTitle%5D)+AND+%22Spine%22%5BJournal%5D)">Epidemiology, demographics, and pathophysiology of acute spinal cord injury.</a> Spine. 26 (24), 2-12 (2001).</li>
<li>Gorio, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recombinant+human+erythropoietin+counteracts+secondary+injury+and+markedly+enhances+neurological+recovery+from+experimental+spinal+cord+trauma%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma.</a> Proc Natl Acad Sci U S A. 99 (14), 9450-9455 (2002).</li>
<li>Windle, W. F., Clemente, C. D., Chambers, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+formation+of+a+glial+barrier+as+a+means+of+permitting+a+peripheral+nerve+to+grow+into+the+brain%5BTitle%5D)+AND+%22J+Comp+Neurol%22%5BJournal%5D)">Inhibition of formation of a glial barrier as a means of permitting a peripheral nerve to grow into the brain.</a> J Comp Neurol. 96 (2), 359-369 (1952).</li>
<li>Young, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spinal+cord+contusion+models%5BTitle%5D)+AND+%22Prog+Brain+Res%22%5BJournal%5D)">Spinal cord contusion models.</a> Prog Brain Res. 137, 231-255 (2002).</li>
<li>Stokes, B. T., Noyes, D. H., Behrmann, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+electromechanical+spinal+injury+device+with+dynamic+sensitivity%5BTitle%5D)+AND+%22J+Neurotrauma%22%5BJournal%5D)">An electromechanical spinal injury device with dynamic sensitivity.</a> J Neurotrauma. 9 (3), 187-195 (1992).</li>
<li>Jakeman, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Traumatic+spinal+cord+injury+produced+controlled+contusion+in+mouse%5BTitle%5D)+AND+%22J+Neurotrauma%22%5BJournal%5D)">Traumatic spinal cord injury produced controlled contusion in mouse.</a> J Neurotrauma. 17 (4), 299-319 (2000).</li>
<li>Sahni, V., Kessler, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem+cell+therapies+for+spinal+cord+injury%5BTitle%5D)+AND+%22Nat+Rev+Neurol%22%5BJournal%5D)">Stem cell therapies for spinal cord injury.</a> Nat Rev Neurol. 6, 363-372 (2010).</li>
<li>Marfia, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adult+neural+precursors+isolated+from+post+mortem+brain+yield+mostly+neurons%3A+an+erythropoietin-dependent+process%5BTitle%5D)+AND+%22Neurobiol+Dis%22%5BJournal%5D)">Adult neural precursors isolated from post mortem brain yield mostly neurons: an erythropoietin-dependent process.</a> Neurobiol Dis. 43 (1), 86-98 (2011).</li>
<li>Gritti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multipotent+neural+stem+cells+reside+into+the+rostral+extension+and+olfactory+bulb+of+adult+rodents%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Multipotent neural stem cells reside into the rostral extension and olfactory bulb of adult rodents.</a> J Neurosci. 22 (2), 437-445 (2002).</li>
<li>Whetstone, W. D., Hsu, J. Y., Eisenberg, M., Werb, Z., Noble-Haeusslein, L. J. J Neurosci Res. 74 (2), 227-239 (2003).</li>
<li>Gonzalez-Lara, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+cellular+magnetic+resonance+imaging+to+track+the+fate+of+iron-labeled+multipotent+stromal+cells+after+direct+transplantation+in+a+mouse+model+of+spinal+cord+injury%5BTitle%5D)+AND+%22Mol+Imaging+Biol%22%5BJournal%5D)">The use of cellular magnetic resonance imaging to track the fate of iron-labeled multipotent stromal cells after direct transplantation in a mouse model of spinal cord injury.</a> Mol Imaging Biol. 13 (4), 702-711 (2010).</li>
<li>Ottobrini, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Magnetic+resonance+imaging+of+stem+cell+transplantation+in+injured+mouse+spinal+cord%5BTitle%5D)+AND+%22Cell+R4%22%5BJournal%5D)">Magnetic resonance imaging of stem cell transplantation in injured mouse spinal cord.</a> Cell R4. 2 (3), e963(2014).</li>
<li>Janowwski, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurotransplantation+in+mice%3A+The+concorde-like+position+ensures+minimal+cell+leakage+and+widespread+distribution+of+cells+transplanted+into+the+cistern+magna%5BTitle%5D)+AND+%22Neuroscience+Letter%22%5BJournal%5D)">Neurotransplantation in mice: The concorde-like position ensures minimal cell leakage and widespread distribution of cells transplanted into the cistern magna.</a> Neuroscience Letter. 430 (2), 169-174 (2008).</li>
<li>Basso, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Basso+Mouse+Scale+for+locomotion+detects+differences+in+recovery+after+spinal+cord+injury+in+five+common+mouse+strains%5BTitle%5D)+AND+%22J+Neurotrauma%22%5BJournal%5D)">Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains.</a> J Neurotrauma. 23 (5), 635-659 (2006).</li>
<li>Hofstetter, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Allodynia+limits+the+usefulness+of+intraspinal+neural+stem+cell+grafts%3B+directed+differentiation+improves+outcome%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Allodynia limits the usefulness of intraspinal neural stem cell grafts; directed differentiation improves outcome.</a> Nat Neurosci. 8 (3), 346-353 (2005).</li>
<li>Bottai, D., Madaschi, L., Di Giulio, A. M., Gorio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Viability-dependent+promoting+action+of+adult+neural+precursors+in+spinal+cord+injury%5BTitle%5D)+AND+%22Mol.+Med%22%5BJournal%5D)">Viability-dependent promoting action of adult neural precursors in spinal cord injury.</a> Mol. Med. 14 (9-10), 634-644 (2008).</li>
<li>Bottai, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Embryonic+stem+cells+promote+motor+recovery+and+affect+inflammatorycell+infiltration+in+spinal+cord+injured+mice%5BTitle%5D)+AND+%22Exp+Neurol%22%5BJournal%5D)">Embryonic stem cells promote motor recovery and affect inflammatorycell infiltration in spinal cord injured mice.</a> Exp Neurol. 223 (2), 452-463 (2010).</li>
</ol>

The authors declare no competing financial interest.

In this paper we described a method to obtain a reproducible model of traumatic spinal cord injury using an Infinite Horizon Impactor at a force of 70 kdyne (severe). Using a larger force paradigm (80&#160;kdyne), we can cause a more severe injury that unfortunately is associated with higher mice mortality. In order to avoid this problem, we commonly choose a moderate force paradigm (70 kdyne) that is associated to a repeatable lesion with a gradual recovery of function and lower mortality. To produce such a stable injur...

A spinal cord injury (SCI) is the most common injury caused by high-energy trauma like motor vehicles accidents, falls, sports and violence 1. In severe SCI, the injury force destroys or damages neural tissue, causing sudden loss of neurological function. Traumatic SCI occurs frequently in young adults between 10 and 40 years of age. It greatly affects the patient&#8217;s mental and physical condition and causes enormous economic impact to society 2. The treatment approach in the acute phase is often limited to a high-dose of corticosteroid, surgical stabilization and decompression to possibly attenuate further damage 3-4, but the roles of these methods on locomotor recovery after SCI are still controversial. In addition to acute tissue loss, the traumatic injury and the activation of secondary mechanisms of degeneration cause demyelination and death of multiple cell types 5-6. The degree of recovery of function can often be correlated to the extent of spared white matter at the injury site 7.
Animal models of SCI may be used both to investigate the biological responses of the tissue to injury and to test potential therapies. Moreover, a useful animal model of a human pathology not only has to reproduce some aspects of that condition but also must offer advantages over direct clinical observation and experiment. The most widely used models of spinal cord injury involve contusion or compression injury delivered to the surgically exposed spinal cord 8. The development of a controlled weight-drop contusion injury represent an important milestone in the history of SCI research. The Ohio State University spinal cord research center has pursued the technological challenge of a device that can be used to induce a particular compression of the spinal cord with parameters of impact controlled by a computer 9. This was originally designed for use with rats; later it was modified to apply towards mice 10. The advantages of this kind of approach are that the biomechanics of injury can be studied more in depth and the parameters of injury can be defined in a more complete manner in order to obtain a reproducible experimental model, therefore allowing more precise evaluation of the effects of tested treatments on the functional recovery process.
Many studies have evaluated the transplantation effects of a variety of stem cells in SCI models 11. We have recently isolated adult neural stem cells from the Sub-Ventricular Zone (SVZ) several hours after death of the mouse donor 12-13. This procedure provides a population of neural stem cells, called post mortem neural precursors (PM-NPCs), which seem to be advantageous in a regenerative medicine approach for curing SCI. In this paper we will demonstrate: i) the protocol for cell labeling with the vital tracer PKH26, ii) the surgical procedure to perform on traumatic SCI, and iii) the intravenous (i.v.) administration of labeled cells. Moreover, in this work we demonstrate that transplanted cells migrate to spinal cord lesion sites and differentiate mostly into microtubule associated protein (MAP) 2 positive cells. Furthermore, the differentiation is accompanied by the promotion of a stable recovery of hind limb function.

<table><tbody><tr><td>PKH26GL-1KT&amp;nbsp;</td><td>Sigma</td><td>091M0973</td><td></td></tr><tr><td>Infinite horizon (IH) Impactor device&amp;nbsp;</td><td>Precision Systems and Instrumentation, LLC</td><td>Model 0400 Serial 0171</td><td></td></tr><tr><td>Gentamycin 10 mg/ml</td><td>Euroclone</td><td>ECM0011B</td><td>1 mg/ml in sterile saline solution</td></tr><tr><td>Isoflurane-Vet 250 ml</td><td>Merial</td><td>B142J12A</td><td></td></tr><tr><td>Blefarolin POM OFT 10 g</td><td></td><td></td><td></td></tr><tr><td>Slide Warmer</td><td>2Biological Instruments</td><td>HB101-sm-402</td><td></td></tr><tr><td>Scalpel, size 10</td><td>Lance Paragon</td><td>26920</td><td></td></tr><tr><td>Small Graefe Forceps</td><td>2Biological Instruments</td><td>11023-14</td><td></td></tr><tr><td>Rongeur</td><td>Medicon Instruments</td><td>07 60 07</td><td></td></tr><tr><td>Micro scissors</td><td>2Biological Instruments</td><td>15000-00</td><td></td></tr><tr><td>Absorbable sutures (4/0)</td><td>Safil Quick</td><td>C0046203</td><td></td></tr><tr><td>Hemostat</td><td>2Biological Instruments</td><td>13014-14</td><td></td></tr><tr><td>Reflex 7 wound clip applicator</td><td>2Biological Instruments</td><td>12031-07</td><td></td></tr><tr><td>7 mm Reflex wound clips</td><td>2Biological Instruments</td><td>12032-07</td><td></td></tr><tr><td>NGS</td><td>Euroclone</td><td>ECS0200D</td><td></td></tr><tr><td>Triton X 100</td><td>Merck Millipore</td><td>1086431000</td><td></td></tr><tr><td>Anti Microtubule Assocoated Protein&amp;nbsp; (MAP) 2</td><td>Millipore</td><td>AB5622</td><td></td></tr><tr><td>Alexa Fluor 488</td><td>Invitrogen</td><td>A11008</td><td></td></tr><tr><td>FluorSave Reagent&amp;nbsp;</td><td>Calbiochem</td><td>345789</td><td></td></tr><tr><td>Neural stem cells medium</td><td></td><td></td><td>DMEM-F12 medium (Euroclone) containing 2 mm l-glutamine (Euroclone), 0.6% glucose (Sigma-Aldrich), 9.6 gm/ml putrescine (Sigma-Aldrich), 6.3 ng/ml progesterone (Sigma-Aldrich), 5.2 ng/ml sodium selenite (Sigma-Aldrich), 0.025 mg/ml insulin (Sigma-Aldrich), 0.1 mg/ml transferrin (Sigma-Aldrich), and 2 &amp;mu;g/ml heparin (sodium salt, grade II; Sigma-Aldrich), bFGF (human recombinant, 10 ng/ml; Life Technologies) and EGF (human recombinant, 20 ng/ml; Life Technologies)&amp;nbsp;</td></tr><tr><td>DMEM-F12</td><td>Euroclone</td><td>ASM5002</td><td></td></tr><tr><td>l-glutamine</td><td>Euroclone</td><td>ECB3000D</td><td></td></tr><tr><td>glucose</td><td>Sigma-Aldrich</td><td>G8270-100G</td><td></td></tr><tr><td>putrescine</td><td>Sigma-Aldrich</td><td>P5780-25G</td><td></td></tr><tr><td>progesterone</td><td>Sigma-Aldrich</td><td>P6149-1MG</td><td></td></tr><tr><td>Sodium-selenite</td><td>Sigma-Aldrich</td><td>S9133-1MG</td><td></td></tr><tr><td>transferrin</td><td>Sigma-Aldrich</td><td>T 5391</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I1882</td><td></td></tr><tr><td>Heparin sodium-salt</td><td>Sigma-Aldrich</td><td>H0200000</td><td></td></tr><tr><td>bFGF</td><td>Life Technology</td><td>PHG0024</td><td></td></tr><tr><td>h-EGF</td><td>Life Technology</td><td>PHG6045</td><td></td></tr><tr><td>Syringe 0.33 cc 29 G</td><td>Terumo</td><td>MYJECTOR&amp;nbsp;</td><td></td></tr><tr><td>buprenorphine</td><td>Schering Plough SpA</td><td>TEMGESIC</td><td></td></tr><tr><td>eye gel</td><td>Bausch &amp;amp; Lomb</td><td>LIPOSIC</td><td></td></tr></tbody></table>

neural stem cell transplantation in experimental contusive model of spinal cord injury

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

The total number of transplanted cells is 1 x 106 cells and was divided into three consecutive injections in the tail vein. We administered 3.3 x 105 cells in 50 µl of phosphate buffer solution (PBS). The first injection was performed within 30 min after injury, the second 6 hr later and the last 18 hr after the lesion. The choice of a time limit of 18 hr after SCI for administering PM-NPCs was determined by the optimal permeability of the blood brain barrier at this time 14. To evaluate ...

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

neural-stem-cell-transplantation-experimental-contusive-model-spinal

Research

JoVE Journal

Medicine

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.

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

Source: Lakshman, N., et al. A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury. J. Vis. Exp. (2018).This video demonstrates the activation and migration of neural stem cells (NSCs) in a mouse spinal cord injury model. To expose the spinal cord, a vertebra from the thoracic region of the spine is removed from an anesthetized mouse. A superficial injury is then created in the white matter of the spinal cord using a bent needle,...

 A Minimal Spinal Cord Injury Model to Study Neural Stem Cell Activation and Migration

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

A Contusion Model of Severe Spinal Cord Injury in Rats

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.

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

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Severe spinal cord injuries often result in tissue defects. Two possibilities are described to successfully bridge such gaps to promote tissue adaptation, regenerative responses and functional improvement in rats via implantation of a mechanical microconnector system after acute injury and five weeks after complete spinal cord transection.

Experimental Strategies to Bridge Large Tissue Gaps in the Injured Spinal Cord after Acute and Chronic Lesion

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.

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

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.

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

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

In this paper, we provide a reproducible method to generate and maintain long-term spinal cord organotypic slices transplanted with neural stem cells as an ex vivo model for testing cellular replacement therapies.

Long-Term Mouse Spinal Cord Organotypic Slice Culture as a Platform for Validating Cell Transplantation in Spinal Cord Injury

In this study, nerve-mimetic composite hydrogels were developed and characterized that can be utilized to investigate and capitalize on the pro-regenerative behavior of adipose-derived stem cells for spinal cord injury repair.

Development of Combinatorial Therapeutics for Spinal Cord Injury using Stem Cell Delivery

Learn neural stem cell transplantation in a mouse spinal cord injury contusion model using PKH 26-labeled precursor cells. This method enables tracking of engrafted cells and studying regeneration potential.

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Explore More Videos