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 transplantat...

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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.

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			<td height="21" style="height:21px;width:287px;">PKH26GL-1KT&#160;</td>
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			<td style="width:119px;">13014-14</td>
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			<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>
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			<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>
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			<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>
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			<td height="21" style="height:21px;width:287px;">Syringe 0.33cc 29G</td>
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			<td height="21" style="height:21px;width:287px;">eye gel</td>
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			<td style="width:119px;">LIPOSIC</td>
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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 vues.  University of Milan.  Lésion de la moelle épinière est une condition traumatique qui entraîne une morbidité grave et une mortalité élevée. Dans ce travail, nous décrivons en détail un modèle de contusion de blessure de la moelle épinière chez la souris suivie d'une transplantation de cellules souches neurales. 