We would like to acknowledge the funding support for this work from the Canadian Institutes of Health Research (CIHR), the Krembil Family Foundation, the Halbert Chair in Neural Repair and Regeneration, Phillip and Peggy DeZwirek, and Gordon Yao for the contribution to Figure 2. Klaus Zweckberger was funded by a grant from the &#8220;Deutsche Forschungsgesellschaft&#8221; (DFG).

NOTE: The following experimental protocol was approved by the animal care committee of the University Health Network (Toronto, Canada) and is in accordance with the policies established in the guide to the care and use of experimental animals prepared by the Canadian council of animal care.
1. Cervical Aneurysm Clip Contusion/Compression Model
<ol>
	<li>Before surgery autoclave instruments and keep sterile conditions during the entire surgcial procedure by putting the instruments in a 70% alcohol bath.</li>
	<li>Anaesthetize Wistar rats (250-270 g) with a combination of oxygen (O2), nitrous oxide (N2O) (1:1), and 1.8-2.2% Isoflurane and support spontaneous breathing via a gas anesthesia mask. For induction of anesthesia start with 5% Isoflurane for 1 minute and reduce afterwards. Before starting surgery, control depth of anesthesia by giving a painful stimulus (e.g. at the paws). Apply fatty ointments in the eyes to avoid dryness and to prevent subsequent infections.</li>
	<li>Put the rats onto a heating cushion (37 &#176;C) and fix the head into a stereotactic frame.</li>
	<li>Shave the surgical region around the cervical spine and disinfect with povidone iodine and 70% alcohol.</li>
	<li>Make a midline incision above the spine from the cervical vertebra (C2) reaching to the prominent processus of the thoracic vertebral body 2 (T2).</li>
	<li>Cut through the outer layer of the vertebral muscles directly on the midline (to avoid bleeding) in a cranio-caudal direction and further dissect the deeper muscle layers bluntly until you reach the processus spinosus and the laminae. Insert retractors.</li>
	<li>Orientate the prominent processus spinosus of the vertebra body T2 to observe the targeted levels for laminectomy. After identification and micro-surgical preparation of the selected laminae, cut through the ligamenti flavae to loosen the laminae and the processus spinosus. Finally, cut through the laminae with a bone clipper lateral to the spinal cord and remove those gently, avoiding any compression of the spinal cord itself. 
	NOTE: Most common levels are C5/6, C6/7, or C7/T1. The perioperative mortality rate increases the more rostral the level of injury. Bleeding from the paravertebral venous sinus is common and can be addressed by careful compression with sponge.</li>
	<li>Before inserting the clip to traumatize the cord, identify emerging nerve roots to spare them from clipping (especially at level C5/6).</li>
	<li>In order to ensure smooth clip induction, loosen the ventral dura from the dorsal side of the vertebral bodies with a hook and prepare a corridor for the clip.</li>
	<li>Finally, insert the open clip and let it snap shut (rapid closure) to achieve a contusion injury. Clip closing force and duration of clip closure determine the intensity of the trauma and the extent of compression. Commonly used are clip forces of between 15-35 g and a clipping duration of e.g. 1 min. (Figure 1).</li>
	<li>After removal of the clip, adapt muscles in 2 layers and close the wound.</li>
	<li>Stop anesthesia and let the animal wake up under your continuous observation until it regains sufficient consciousness for sternal recumbency. Finally, put the rat in a single cage and follow post-operative treatment guidelines.</li>
	<li>Since animals struggle with the severity of this type of injury, you must pay special attention to post-operative treatments:
	<ol>
		<li>Administer painkillers (Buprenorphine and Meloxicam for 3 days and 5 days, respectively and according to the clinical symptoms).</li>
		<li>Give additional saline solution subcutaneously for 3 days (2 times a day, 5-10 milliliter (ml)).</li>
		<li>Provide antibiotics in the drinking water 2 days prior and until 7 days post surgery (e.g. Moxifloxacine)</li>
		<li>Squeeze the urinary bladder 2-3 times a day until recovery of bladder function is constantly apparent.</li>
		<li>Observe neurological deficits and physiological condition of the operated animals at least once a day.</li>
	</ol>
	</li>
</ol>
2. Injecting SAPs and NPCs (14 days after injury)
<ol>
	<li>Induce anesthesia as described in 1.1. to 1.3, fix the head of the rat in a stereotactic frame, remove the stitches or wound clips, and disinfect the wound and the surgical area with povidone iodine and 70% alcohol.</li>
	<li>Carefully dissect the paravertebral muscles, insert retractors, remove scar tissue microscopically from the dura, and re-expose the lesion site.</li>
	<li>Prepare SAPs in a concentration of 1% (w/v) to perform an extracellular matrix gel. QL6 SAPs have a physiologically compatible pH and do not need to be buffered prior to injection. For visualization of SAPs in the spinal cord, use a fluorescent derivate of QL6 (QL6-FITC).</li>
	<li>Inject SAPs (5 microliter (&#956;l) into the center of the lesion, distributed in 2 portions, each 2.5 &#956;l bilateral of the midline. Use a Hamilton syringe connected to the stereotactic frame with a micro glass capillary (100 micrometer (&#956;m) outer diameter (OD)). Open the dura carefully with the tip of a sharp needle, and insert the glass capillary stereotactically 2 millimeter (mm) into the traumatized spinal cord.</li>
	<li>After injecting 1/3 of the volume, remove the needle to 1.5 mm depth, and after a further 1/3 to 1 mm. After injection of the entire volume, and before removal of the syringe, wait 5 minutes to stabilize gel formation.</li>
	<li>To generate NPCs, use adult DsRed mice (or YFP positive mice, green) and isolate and cultivate them from the paraventrical zone4,18,21.</li>
	<li>Assess viability of NPCs by Trypan Blue staining indicating the presence of ~90% live cells in the cell suspension. Dilute the cells in growth medium (50 x 103 live cells/&#956;l) and then use them for cell transplantation.</li>
	<li>Make four 2 &#956;l (8 &#956;l total volume, containing 4 x 105 NPCs) intraspinal injections bilaterally at 2 mm rostral and caudal of the injury site. After opening of the dura, insert the Hamilton micro glass capillary 1.5 mm below the dorsal surface of the spinal cord and inject 2 &#956;l of the cell suspension. Choose an injection rate at about 0.5 &#956;l /min (min).</li>
	<li>At the end of each injection and before removal of the capillary out of the cord, wait for at least 1 min allowing tissue stretching to accommodate the new cell volume. (Figure 2)</li>
</ol>
3. Implantation of Subdural Pumps for Growth Factor Application
<ol>
	<li>In order to enrich the cerebral spinal fluid (CSF) with growth factors supporting cell survival, use micro-osmotic pumps diluting growth factors sub-durally throughout 7-14 days, with a dilution rate of 0.5 &#956;l/hr, a catheter diameter of 0.04 cm OD, and a reservoir volume of 100 &#956;l.</li>
	<li>Choose preferred growth factors (e.g., brain derived growth factor (BDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF)), fill the pumps 6-10 hr prior implantation, and equilibrate pumps in a water bath at 37 &#176;C.</li>
	<li>Immediately after NPC injections, prepare a subcutaneous recess to place the pump. Preferred locations are the lateral flanks of thoraco- abdominal region avoiding major local discomfort caused by the pump itself.</li>
	<li>To gain the highest concentration of growth factors, ensure that the open tip of the catheter ends close to the lesion site. Therefore, perform a skip-laminectomy of the adjacent upper or lower level. For example, if the SCI is at C7/T1, perform a small laminectomy of C5.</li>
	<li>Put the pump into the subcutaneous recess, shorten the catheter to the necessary length, and secure it with several sutures (6.0) on the paravertebral muscles avoiding any movement-associated dislocation.</li>
	<li>After opening the dura (e.g., at C5) with the sharp tip of a needle, introduce the catheter into the subdural space and let it glide in a caudal direction without any resistance and without injuring the cord. The skipped lamina of e.g. C6 serves as additional point of fixation and stabilization of the catheter.</li>
	<li>Make sure that the catheter runs smoothly and does not fold.</li>
	<li>Close muscles by layer, and skin, with sutures or clips. (Figure 3)</li>
	<li>Stop anesthesia and let the animal wake up under your continuous observation until it regains sufficient consciousness for sternal recumbency. Finally, put them back in a single cage and follow post-operative treatment guidelines.</li>
	<li>Provide special postoperative treatment, even though, animals might have recovered partly at this time-point:
	<ol>
		<li>Administer painkillers (Buprenorthine and Meloxicam for 3 days and 5 days, respectively and according to the clinical symptoms).</li>
		<li>Provide antibiotics in water bottles 2 days prior until 7 days post-surgery (e.g. Moxifloxacine).</li>
		<li>Continue squeezing the urinary bladder, if still necessary.</li>
		<li>Give additional subcutaneous fluids, if the rats appear dehydrated.</li>
		<li>Keep observing the neurological function and physiological condition of the operated animals at least once a day.</li>
		<li>Administer immunosuppression treatment 2 days prior NPC injection and until 7 days post transplanation (minocycline) and until sacrifice (sandimmune), respectively.</li>
	</ol>
	</li>
</ol>
4. Tissue Assessment
<ol>
	<li>Sacrifice animals at the end of the observation time (e.g. 4 weeks after SCI) in deep anesthesia (5% Isoflurane for 2-3 min) and perfuse them transcardially with 50 ml cold (4 &#176;C) saline followed by 150 ml cold 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS).</li>
	<li>Remove the spinal cord and put it in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) for 24 hr.</li>
	<li>Put the spinal cords in 25% sucrose solution for cryoprotection for 48 hr.</li>
	<li>Provide longitudinal cryosections with a thickness of &#60;30 &#956;m.</li>
	<li>For immunohistochemical staining of all cell-nuclei providing a background use DAPI (1:1,000). DsRed positive NPCs appear red, QL-6 FITC appears green, and both thus need not to be stained specially. (Figure 4).</li>
	<li>For scanning electron microscopy (SEM) let samples soak in glutaraldehyde at 4 &#176;C for 2 hr, dehydrate them slowly in 10% increment steps of ethanol for 5 min and place them in a pressurized liquid CO2 siphon for 1 hr. Coat scaffolds with gold using a sputter coater. Take images with a Hitachi S-3400N scanning electron microscope. (Figure 5)</li>
</ol>

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Covidien supplied the QL6 SAP for this study.

This protocol has been developed to enable the reader to perform a cervical injury model in rats and to use a combined treatment approach with SAPs and NPCs promoting better recovery after cervical SCI.
Particularly in comparison to other cervical trauma models, such as the (hemi)-transection model or the weight drop and concussion models, the clip contusion/compression model represents both major pathophysiological trauma mechanisms &#8211; contusion and compression- and therefore mimics best clinical conditions. While it is well established for the use in rat and mouse thoracic spine7-11, it has recently been adapted for use in the cervical spine. This was an important step since cervical spinal cord injuries are the most frequently seen in the clinic and cervical anatomy varies significantly from the thoracic spine. Furthermore, cervical SCI patients suffering from tetraparesis are keen to regain at least partial motor function of their upper extremities.
Challenging aspects of an experimental cervical SCI, however, are a high mortality rate that increases in a rostral direction from C7 to C5 and can reach up to 20-30% in the middle and upper cervical spine. Furthermore, emerging nerve roots (especially at C5/6) are sensitive to manipulation, and in the case they are not preserved, damage and irritation might cause chewing of the frontal paws resulting in the necessity of an early sacrifice of the animals. In general, animals with cervical spinal cord injury need a lot of post-operative attention and care and show a longer course of recovery. On the other hand, in addition to careful surgical preparation, these issues can partly be addressed by adapting the severity of the injury by selecting an appropriate clip closing force (e.g. 15 to 35 g) and an adequate clipping time (e.g. 1 min). Modifying these parameters lead to different severities of injury (mild, moderate, or severe SCI). Furthermore, there might be a risk of surgeon dependency or an unequal distribution of the damage. To address these issues the use of a clip applicator is an option where the clip always is released and therefore closed with the same speed and hence velocity. Before snapping the clip it is mandatory to ensure that the clip has its correct position and encloses the entire spinal cord equally. Special surgical challenge lies in the re-exposure of the lesion site removing the scar tissue from the dura. It is recommended that this procedure is executed micro-surgically, using sharp preparation, and avoiding any pressure or pulling of the fixed cord.
Self-assembling peptides have been identified having potentials to bridge the cavity and, furthermore, to improve the inhibitory environment at the lesion site finally even leading to axonal regeneration and sprouting5,12,13,14. QL-6 nanofibers assemble to scaffolds bridging the intramedullary cavity and, thus, can provide a matrix for axonal sprouting and regeneration and improve the inhibitory environment before cell transplantation. The advantages of these peptides lay in their low viscosity (fluid) and the neutral pH. Especially in comparison to high-viscous hydrogels or tissue scaffolds, SAPs can easily injected into the injured spinal cord and additional injuries derived from the injection itself are limited.
Although QL6 nanofibers themselves might improve cell survival4,15, nevertheless, the use of growth factors seems to be advantageous16,17,18,19,20,21. They can be administered either by hydrogels releasing growth factors22, or by application via osmotic pumps (as described above). Osmotic pumps connected to subdural catheters offer the advantage of a continuous and controlled release and thus, enrichment of the cervical spinal fluid (CSF). Placing the subdural catheter, however, is challenging especially with respect to vast scarring that might have occurred after some weeks post injury.
In order to provide good tissue conditions for NPC survival and integration, several studies have chosen injection points in the adjacent white matter, 2 mm rostral or caudal to the lesion site and not directly in the epicenter of the lesion4,15,18. There, NPCs have a better chance for survival, integration, and differentiation into astrocytes, oligodendrocytes or neurons, and can migrate towards or into the lesion resulting in axonal sprouting and improved axonal connectivity.
After mastering these techniques this proposed protocol might to be adopted by the individual users according their necessities and interests encompassing modifications of the level and severity of the injury, the use of different cell types or growth factors, or the addition of other neuro-protective substances.
In summary, combined treatment with SAPs and NPCs might offer a novel and promising approach overcoming the most challenging obstacles in the treatment of SCI: cavities and tissue scarring.

More than 50% of spinal cord injuries are related to the cervical spine. In the clinical setting two major pathophysiological mechanisms are described: the initial contusion of the spinal cord and subsequently, the ongoing compression caused by bone fractures, hemorrhages or tissue swelling.
The aneurysm clip contusion/compression model mimics both pathophysiological mechanisms: snapping the clip produces a contusion and the duration of clipping represents the compression component, conceding that the compression in clinical settings caused by bone fractures, hemorrhages or tissue swelling last significant longer. The used aneurysm clip is modified by a ring spring guaranteeing exact and reproducible clipping force. Especially in comparison to the hemi-transection or the contusion model, this aneurysm clip model mimics best clinical settings. While patients with thoracic injuries suffer from paraplegia, most patients with cervical injuries are tetraplegic and completely dependent. The anatomical structure of the cervical cord, however, shows significant differences compared to the thoracic or lumbar spine, and thus, is addressed in particular in this protocol.
The development of intramedullary cavities and tissue scarring are obstacles for recovery and regeneration. To overcome these barriers the use of scaffold material is a promising approach. Self-assembling peptides can be injected directly into the epicenter of the lesion. There they assemble into nano-fiber scaffolds bridging the cavity and improve the inhibitory environment by reducing inflammation and tissue scaring. While rigid materials cause considerable damage of the spinal cord during implantation, the fluid peptides can be injected safely and without severe additional damage.
Improving the inhibitory environment with self-assembling peptides before stem cell transplantation, hence, support cell integration, differentiation and finally, functional recovery, after cervical spinal cord injury.

<table><tbody><tr><td>Aneurysmal clip</td><td>SharpTech</td><td></td><td></td></tr><tr><td>Surgical microscope</td><td>Leica</td><td></td><td></td></tr><tr><td>Micro injection system</td><td>World Precision Instruments, Inc.</td><td></td><td></td></tr><tr><td>Small animal stereotaxic instrument</td><td>David Kopf Instruments</td><td></td><td></td></tr><tr><td>Hamilton syringe</td><td>Hamilton company</td><td></td><td></td></tr><tr><td>Subdural pumps</td><td><a href="http://www.alzet.com" target="_blank"><img src="/img/new_window.png" /> Alzet osmotic micro pump 1007D</a></td><td></td><td></td></tr><tr><td>Surgical instrument</td><td>Fine Science tools</td><td></td><td></td></tr><tr><td>Isoflurane USP</td><td>Pharmaceutical Partners of Canada Inc.</td><td></td><td></td></tr><tr><td>0.9% Sodium Chloride injection USP</td><td>Baxter</td><td></td><td></td></tr><tr><td>7.5% Povidone iodine</td><td>Purdue Pharma</td><td></td><td></td></tr><tr><td>70% Isopropyl alcohol USP</td><td>GreenField Ethanol Inc.</td><td></td><td></td></tr><tr><td>QL6 SAP</td><td>Covidien</td><td></td><td></td></tr><tr><td>0.4% Trypan blue</td><td>Gibco</td><td></td><td></td></tr><tr><td>Platelet-Derived Growth Factor (PDGF)</td><td>Sigma</td><td></td><td></td></tr><tr><td>Epidermal Growth Factor (EGF)</td><td>Sigma</td><td></td><td></td></tr><tr><td>Fibroblast Growth Factor (FGF)</td><td>Sigma</td><td></td><td></td></tr></tbody></table>

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.

When performing the above-described procedure, you will get a SAP scaffold bridging the cavity and offering an improvement of the inhibitory environment, less tissue scaring and an increase in NPC survival. Figure 4 shows a longitudinal section of a rat spinal cord obtained at the injury site 6 weeks after SCI and 4 weeks after QL6 SAP injection and NPC transplantation. QL6 peptides were successfully injected into the cord, aggregated in the epicenter and diffused rostro-caudally in the penumbra. Electron microscope imaging further shows, in Figure 5, the assembly of 1% (w/v) QL6 peptides to a nanofiber scaffold within 2 hr diluted in PBS solutions.
This matrix provides an improvement of the inhibitory environment contributing to increased cell survival and cell differentiation, less tissue scarring and finally leads to a better chance for functional recovery.
<img alt="Figure 1" src="/files/ftp_upload/52105/52105fig1highres.jpg" /> 
Figure 1: Clip contusion/compression aneurysm model. (A) Photograph through the surgical microscope after laminectomy of C7/T1 and clip contusion/compression of the spinal cord of a rat. (B) Picture of the clip with the ring spring guaranteeing precise closing force.
<img alt="Figure 2" src="/files/ftp_upload/52105/52105fig2highres.jpg" /> 
Figure 2: Injection points of SAPs and stem cells. Graphical illustration of the injection points: 2 stereotactically performed SAP injections in the epicenter of the lesion, followed by 4 injections of NPCs in the adjacent dorsal columns with a distance of 2 mm caudal and rostral from the epicenter.
<img alt="Figure 3" src="/files/ftp_upload/52105/52105fig3highres.jpg" /> 
Figure 3: Subdural catheter for growth factor administration. Photograph through the surgical microscope: (A) Implanted subdural catheter in order to administer growth factors: the catheter is fixed by several 6.0 sutures at the paraspinal muscles; small opening of the dura at C5; subdural positioning of the catheter with the open catheter end close to the lesion site at C7/T1. (B) Catheter connected to a micro pump placed in a subdural recess on the lateral flank.
<img alt="Figure 4" src="/files/ftp_upload/52105/52105fig4highres.jpg" /> 
Figure 4: Successful delivery of SAPs and NPCs into the cervical spinal cord. Fluorescent staining (DAPI background, blue) of a longitudinal section of a traumatized spinal cord of a rat. Labeled SAPs (QL6-FITC, green) have aggregated in the epicenter of the lesion. Injected NPCs (DsRed positive, red) have diffused in rostral and caudal directions. Scale bar refers to 5 mm.
<img alt="Figure 5" src="/files/ftp_upload/52105/52105fig5highres.jpg" /> 
Figure 5: Nanofiber scaffold formation. Scanning electron microscope (SEM) image showing nanofiber scaffold formation of assembled SAPs. Scale bar refers to 1 &#956;m).

Watch this Scientific Journal Video about Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury at JoVE.com

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical 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.

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

synergetic-use-neural-precursor-cells-self-assembling-peptides

Nanyang Technological University

Research

JoVE Journal

Medicine

9.2K Views.  University Health Network, Krembil Neuroscience Centre. 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. 

Video: Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical 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) ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Bioengineering

Combining Peripheral Nerve Grafting and Matrix Modulation to Repair the Injured Rat Spinal Cord

Traumatic injury to the spinal cord (SCI) causes death of neurons, disruption of motor and sensory nerve fiber (axon) pathways and disruption of communication with the brain. One of the goals of our research is to promote axon regeneration to restore connectivity across the lesion site. To accomplish this we developed a peripheral nerve (PN) grafting technique where segments of sciatic nerve are either placed directly between the damaged ends of the spinal cord or are used to form a bridge across the lesion. There are several advantages to this approach compared to transplantation of other neural tissues; regenerating axons can be directed towards a specific target area, the number and source of regenerating axons is easily determined by tracing techniques, the graft can be used for electrophysiological experiments to measure functional recovery associated with axons in the graft, and it is possible to use an autologous nerve to reduce the possibility of graft rejection. In our lab we have performed both autologous (donor and recipient are the same animal) and heterologous (donor and recipient are different animals) grafts with comparable results. This approach has been used successfully in both acute and chronic injury situations. Regenerated axons that reach the distal end of the PN graft often fail to extend back into the spinal cord, so we use microinjections of chondroitinase to degrade inhibitory molecules associated with the scar tissue surrounding the area of SCI. At the same time we have found that providing exogenous growth and trophic molecules encourages longer distance axonal regrowth into the spinal cord. Several months after transplantation we perform a variety of anatomical, behavioral and electrophysiological tests to evaluate the recovery of function in our spinal cord injured animals. This experimental approach has been used successfully in several spinal cord injury models, at different levels of injury and in different species (mouse, rat and cat). Importantly, the peripheral nerve grafting approach is effective in promoting regeneration by acute and chronically injured neurons.

Traumatic injury to the spinal cord disrupts communication with the brain. To restore lost connectivity we utilize a peripheral nerve graft to provide a substratum for regenerating fibers in combination with neurotrophic factors and matrix-modulating enzymes to remove inhibitory molecules to promote long distance growth.

Traumatic injury to the spinal cord (SCI) causes death of neurons, disruption of motor and sensory nerve fiber (axon) pathways and disruption of ...

Biology

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

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

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

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

Neuroscience

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.

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 ...

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

After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the spinal cord, tumor resections, or tissue defects resulting from traumatic accidents can aid in facilitating general tissue repair as well as regenerative growth of nerve fibers into and beyond the affected area. Two experimental treatment strategies are presented: (1) implantation of a novel microconnector device into an acutely and completely transected thoracic rat spinal cord to readapt severed spinal cord tissue stumps, and (2) polyethylene glycol filling of the SCI site in chronically lesioned rats after scar resection. The chronic spinal cord lesion in this model is a complete spinal cord transection which was inflicted 5 weeks before treatment. Both methods have recently achieved very promising outcomes and promoted axonal regrowth, beneficial cellular invasion and functional improvements in rodent models of spinal cord injury.
The mechanical microconnector system (mMS) is a multi-channel system composed of polymethylmethacrylate (PMMA) with an outlet tubing system to apply negative pressure to the mMS lumen thus pulling the spinal cord stumps into the honeycomb-structured holes. After its implantation into the 1 mm tissue gap the tissue is sucked into the device. Furthermore, the inner walls of the mMS are microstructured for better tissue adhesion.
In the case of the chronic spinal cord injury approach, spinal cord tissue - including the scar-filled lesion area - is resected over an area of 4 mm in length. After the microsurgical scar resection the resulting cavity is filled with polyethylene glycol (PEG 600) which was found to provide an excellent substratum for cellular invasion, revascularization, axonal regeneration and even compact remyelination in vivo.

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.

After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

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

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord injury. The complete transection model, although not as clinically relevant as the contusion model, is the most rigorous method to evaluate axon regeneration. In the contusion model, it is difficult to distinguish regenerated from sprouted or spared axons due to the presence of remaining tissue post injury. In the complete transection model, a bridging method is necessary to fill the gap and create continuity from the rostral to the caudal stumps in order to evaluate the effectiveness of the treatments. A reliable bridging surgery is essential to test outcome measures by reducing the variability due to the surgical method. The protocols described here are used to prepare Schwann cells (SCs) and conduits prior to transplantation, complete transection of the spinal cord at thoracic level 8 (T8), insert the conduit, and transplant SCs into the conduit. This approach also uses in situ gelling of an injectable basement membrane matrix with SC transplantation that allows improved axon growth across the rostral and caudal interfaces with the host tissue.

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.

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord ...

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

Traumatic spinal cord injury (SCI) induces permanent sensorimotor deficit below the site of injury. It affects approximately a quarter million people in the US, and it represents an immeasurable public health concern. Research has been conducted to provide effective therapy; however, SCI is still considered incurable due to the complex nature of the injury site. A variety of strategies, including drug delivery, cell transplantation, and injectable biomaterials, are investigated, but one strategy alone limits their efficacy for regeneration. As such, combinatorial therapies have recently gained attention that can target multifaceted features of the injury. It has been shown that extracellular matrices (ECM) may increase the efficacy of cell transplantation for SCI. To this end, 3D hydrogels consisting of decellularized spinal cords (dSCs) and sciatic nerves (dSNs) were developed at different ratios and characterized. Histological analysis of dSCs and dSNs confirmed the removal of cellular and nuclear components, and native tissue architectures were retained after decellularization. Afterward, composite hydrogels were created at different volumetric ratios and subjected to analyses of turbidity gelation kinetics, mechanical properties, and embedded human adipose-derived stem cell (hASC) viability. No significant differences in mechanical properties were found among the different ratios of hydrogels and decellularized spinal cord matrices. Human ASCs embedded in the gels remained viable throughout the 14-day culture. This study provides a means of generating tissue-engineered combinatorial hydrogels that present nerve-specific ECM and pro-regenerative mesenchymal stem cells. This platform can provide new insights into neuro-regenerative strategies after SCI with future investigations.

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.

Traumatic spinal cord injury (SCI) induces permanent sensorimotor deficit below the site of injury. It affects approximately a quarter million people in ...

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

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