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>

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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 border="0" cellpadding="0" cellspacing="0" width="743">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Name of Material/ Equipment</td>
			<td style="width:115px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:223px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">PKH26GL-1KT&#160;</td>
			<td style="width:115px;">Sigma</td>
			<td style="width:119px;">091M0973</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Infinite horizon (IH) Impactor device&#160;</td>
			<td style="width:115px;">Precision Systems and Instrumentation, LLC</td>
			<td style="width:119px;">Model 0400 Serial 0171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Gentamycin 10mg/ml</td>
			<td style="width:115px;">Euroclone</td>
			<td style="width:119px;">ECM0011B</td>
			<td>1mg/ml in sterile saline solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Isoflurane-Vet 250ml</td>
			<td style="width:115px;">Merial</td>
			<td style="width:119px;">B142J12A</td>
			<td></td>
		</tr>
		<tr height="21">
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		<tr height="21">
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			<td style="width:115px;">2Biological Instruments</td>
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		<tr height="42">
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			<td style="width:115px;">Medicon Instruments</td>
			<td style="width:119px;">07 60 07</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Micro scissors</td>
			<td style="width:115px;">2Biological Instruments</td>
			<td style="width:119px;">15000-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Absorbable sutures (4/0)</td>
			<td style="width:115px;">Safil Quick</td>
			<td style="width:119px;">C0046203</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hemostat</td>
			<td style="width:115px;">2Biological Instruments</td>
			<td style="width:119px;">13014-14</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Reflex 7 wound clip applicator</td>
			<td style="width:115px;">2Biological Instruments</td>
			<td style="width:119px;">12031-07</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">7mm Reflex wound clips</td>
			<td style="width:115px;">2Biological Instruments</td>
			<td style="width:119px;">12032-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">NGS</td>
			<td style="width:115px;">Euroclone</td>
			<td>ECS0200D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Triton X 100</td>
			<td style="width:115px;">Merck Millipore</td>
			<td style="width:119px;">1086431000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Anti Microtubule Assocoated Protein&#160; (MAP) 2</td>
			<td style="width:115px;">Millipore</td>
			<td style="width:119px;">AB5622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488</td>
			<td style="width:115px;">Invitrogen</td>
			<td style="width:119px;">A11008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FluorSave Reagent&#160;</td>
			<td style="width:115px;">Calbiochem</td>
			<td style="width:119px;">345789</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Neural stem cells medium</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></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 &#956;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)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">DMEM-F12</td>
			<td style="width:115px;">Euroclone</td>
			<td style="width:119px;">ASM5002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">l-glutamine</td>
			<td style="width:115px;">Euroclone</td>
			<td style="width:119px;">ECB3000D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">glucose</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8270-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">putrescine</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5780-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">progesterone</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6149-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Sodium-selenite</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9133-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">transferrin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">T 5391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Insulin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">I1882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Heparin sodium-salt</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">H0200000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">bFGF</td>
			<td style="width:115px;">Life Technology</td>
			<td style="width:119px;">PHG0024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">h-EGF</td>
			<td style="width:115px;">Life Technology</td>
			<td style="width:119px;">PHG6045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Syringe 0.33cc 29G</td>
			<td style="width:115px;">Terumo</td>
			<td style="width:119px;">MYJECTOR&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">buprenorphine</td>
			<td style="width:115px;">Schering Plough SpA</td>
			<td style="width:119px;">TEMGESIC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">eye gel</td>
			<td style="width:115px;">Bausch &#38; Lomb</td>
			<td style="width:119px;">LIPOSIC</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 &#181;l 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 eval...

Watch this Scientific Journal Video about Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury at JoVE.com

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

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 ...

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12.8K Views.  University of Milan. 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.

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

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.

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) ...

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

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.

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

A Novel Vertebral Stabilization Method for Producing  Contusive Spinal Cord Injury

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing reliable cervical SCI is difficult due to lack of satisfactory methods of vertebral stabilization. The conventional method to stabilize the spine is to suspend the rostral and caudal cervical spine via clamps attached to cervical spinous processes. &#160;However, this method of stabilization fails to prevent tissue yielding during the contusion as the cervical spinal processes are too short to be effectively secured by the clamps (Figure 1). &#160;Here we introduce a new method to completely stabilize the cervical vertebra at the same level of the impact injury. &#160;This method effectively minimizes movement of the spinal column at the site of impact, which greatly improves the production of consistent SCIs. &#160;We provide visual description of the equipment (Figure 2-4), methods, and a step-by-step protocol for the stabilization of the cervical 5 vertebra (C5) of adult rats, to perform laminectomy (Figure 5) and produce a contusive SCI thereafter. &#160;Although we only demonstrate a cervical hemi-contusion using the NYU/MASCIS impactor device, this vertebral stabilization technique can be applied to other regions of the spinal cord, or be adapted to other SCI devices. &#160;Improving spinal cord exposure and fixation through vertebral stabilization may be valuable for producing consistent and reliable injuries to the spinal cord. &#160;This vertebral stabilization method can also be used for stereotactic injections of cells and tracers, and for imaging using two-photon microscopy in various neurobiological studies.

Vertebral stabilization is necessary for minimizing variability, and for producing consistent experimental spinal cord injuries.&#160; Using a customized stabilizing apparatus in conjunction with the NYU/MASCIS impactor device, we have demonstrated here the proper equipment and procedure for generating reproducible hemi-contusive cervical (C5) spinal cord injuries in adult rats.

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

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

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

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.

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...