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...

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 border="0" cellpadding="0" cellspacing="0" width="541">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Name</td>
			<td style="width:268px;">Company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aneurysmal clip</td>
			<td>SharpTech</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical microscope</td>
			<td>Leica</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro injection system</td>
			<td>World Precision Instruments, Inc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small animal stereotaxic instrument</td>
			<td>David Kopf Instruments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hamilton syringe</td>
			<td>Hamilton company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Subdural pumps</td>
			<td>Alzet osmotic micro pump 1007D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical instrument</td>
			<td>Fine Science tools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane USP</td>
			<td>Pharmaceutical Partners of Canada Inc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.9% Sodium Chloride injection USP</td>
			<td>Baxter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7.5% Povidone iodine</td>
			<td>Purdue Pharma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Isopropyl alcohol USP</td>
			<td>GreenField Ethanol Inc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QL6 SAP</td>
			<td>Covidien</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.4% Trypan blue</td>
			<td>Gibco</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platelet-Derived Growth Factor (PDGF)</td>
			<td>Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epidermal Growth Factor (EGF)</td>
			<td>Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibroblast Growth Factor (FGF)</td>
			<td>Sigma</td>
		</tr>
	</tbody>
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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. Electro...

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

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JoVE Journal

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9.2K 조회수.  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. 

비디오: 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) ...

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 ...

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

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 ...

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination is disrupted by spinal cord injury (SCI), control of respiratory muscles innervated below the injury level is compromised1,2 leading to respiratory muscle dysfunction and pulmonary complications. These conditions are among the leading causes of death in patients with SCI3. Standard pulmonary function tests that assess respiratory motor function include spirometrical and maximum airway pressure outcomes: Forced Vital Capacity (FVC), Forced Expiratory Volume in one second (FEV1), Maximal Inspiratory Pressure (PImax) and Maximal Expiratory Pressure (PEmax)4,5. These values provide indirect measurements of respiratory muscle performance6. In clinical practice and research, a surface electromyography (sEMG) recorded from respiratory muscles can be used to assess respiratory motor function and help to diagnose neuromuscular pathology. However, variability in the sEMG amplitude inhibits efforts to develop objective and direct measures of respiratory motor function6. Based on a multi-muscle sEMG approach to characterize motor control of limb muscles7, known as the voluntary response index (VRI)8, we developed an analytical tool to characterize respiratory motor control directly from sEMG data recorded from multiple respiratory muscles during the voluntary respiratory tasks. We have termed this the Respiratory Motor Control Assessment (RMCA)9. This vector analysis method quantifies the amount and distribution of activity across muscles and presents it in the form of an index that relates the degree to which sEMG output within a test-subject resembles that from a group of healthy (non-injured) controls. The resulting index value has been shown to have high face validity, sensitivity and specificity9-11. We showed previously9 that the RMCA outcomes significantly correlate with levels of SCI and pulmonary function measures. We are presenting here the method to quantitatively compare post-spinal cord injury respiratory multi-muscle activation patterns to those of healthy individuals.

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment RMCA in Individuals with Chronic Spinal Cord Injury

The purpose of this publication is to present our original work on a multi-muscle surface electromyographic approach to quantitatively characterize respiratory muscle activation patterns in individuals with chronic spinal cord injury using vector-based analysis.

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination ...

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.

Respiratory failure is the leading cause of death following a cervical spinal cord injury. Having a reproducible, quantifiable, and reliable pre-clinical animal model of respiratory failure induced by a partial cervical injury will help to understand the subsequent respiratory and non-respiratory neuroplasticity and allow testing putative repair strategies.

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed ...

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 ...

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 ...