German Legal Casualty Insurance (DGUV), Research Commission of the Medical Faculty of the Heinrich-Heine-University

Institutional guidelines for animal safety and comfort were adhered to, and all surgical interventions and pre- and post-surgical animal care were provided in compliance with the German Animal Protection law (State Office, Environmental and Consumer Protection of North Rhine- Westphalia, LANUV NRW).
1. Complete Transection of the Thoracic Spinal Cord of Female Wistar Rats (220 - 250 g)
<ol>
	<li>Preparation of the Spinal Cord
	<ol>
		<li>Use isoflurane inhalation anesthesia (2 - 3% of isoflurane in O2/NO2 at a ratio of 1:2) and bolus injection of Carprofen (subcutaneously [s.c.] 5 mg/kg). The combination of carprofen and the opioid buprenorphine (0.02 mg/kg s.c.) is recommended. Start surgery when eye lid reflex to light touch with a cotton bud and paw withdrawal reflex to pinching stimulus with forceps are no longer observed.</li>
		<li>Place the animal on a heating blanket at 37 &#176;C to maintain body temperature during surgery and put eye ointment on eyes to avoid dryness while under anaesthesia.</li>
		<li>Shave the animal&#39;s back and prepare the skin with a skin disinfectant.</li>
		<li>Cut the skin at the midline along the thoracic vertebrae for 4 cm with a surgical blade and open it. From this step on, use sterilized surgical instruments for all procedures (autoclaved or immersion-sterilized).</li>
		<li>Retract the muscles above the thoracic vertebrae using a small muscles clamp.</li>
		<li>Remove the spinous processes at thoracic level 8 (Th8) and Th9 with a bone rongeur by cautiously clipping small pieces of bone until the vertebral bones are flat.</li>
		<li>Use anatomical forceps to lift the vertebral column at spinous process Th7 and use a rongeur to clip small pieces of vertebral bone from caudal to rostral until a laminectomy is performed at Th8 and Th9. Expose the dura mater without damaging it by carefully removing only few and very small pieces of vertebral bone at a time.</li>
		<li>Clamp the backbone by 2 stabilization clamps at spinous processes Th7 and Th10, lift the animal to uncouple breathing movements from the vertebrae.</li>
	</ol>
	</li>
	<li>Complete Spinal Cord Transection at Thoracic Level 8/9
	<ol>
		<li>Lift the dura mater with fine forceps, cut the dura mater with fine eye scissors in&#160;transversal direction.</li>
		<li>Hold the lateral cut end of the dura mater with fine forceps and insert a spinal hook into the subarachnoidic space between dura mater and arachnoidea. Avoid damage of the meninges by not pricking into the pia or dura mater with either forceps or spinal cord hook.</li>
		<li>Slowly rotate the hook to place it all along the spinal cord tissue, taking care not to prick into the dura (pia is still intact).</li>
		<li>Lift the spinal cord for approximately 1 - 2 mm upwards, until a gap is seen at the ventral side between spinal cord tissue and dura.</li>
		<li>Insert fine eye scissors into the space between dura and pia and cut the spinal cord while the spinal hook is left in place.</li>
		<li>Lift the two stumps of the spinal cord with two forceps and visually ensure complete transection.</li>
		<li>For control-lesioned animals and animals with a chronic injury, close the dura by interrupted&#160;sutures with monofilament non-adsorbable 9.0 threads.</li>
		<li>For chronic lesions follow part 1.6.</li>
	</ol>
	</li>
	<li>mMS Implantation
	<ol>
		<li>Place mMS above the injury site with the two tubes lying on each lateral side of the vertebra and lower it into lesion cavity.</li>
		<li>Suture tubes to the muscles at the side of the vertebra with non-resorbable 4-0 thread to ensure stabilization of the mMS. Ensure a fixation of the mMS using forceps during this step.</li>
		<li>Remove the mMS connector pin by cutting with a pair of fine scissors.</li>
		<li>Close the dura above the mMS and suture it with 9.0 threads.</li>
		<li>Attach one tube to the vacuum pump, and seal the other by clamping.</li>
		<li>Apply gentle negative pressure to the mMS by a vacuum pump via the open tube, sucking the spinal cord stumps into the mMS lumen. Apply the negative pressure for several minutes (at maximum for 10 min) and monitor by sensors (250 - 350 mbar).</li>
		<li>Cut the tubes close to the mMS and remove the tubes. Continue with Step 1.6.</li>
	</ol>
	</li>
	<li>Resection of Spinal Cord Tissue Including the Chronic Lesion Scar at Week 5 after Initial Injury
	<ol>
		<li>Follow steps 1.1.1 - 1.1.5.</li>
		<li>Identify scar tissue by brown-yellow appearance and stiff tissue on top of the spinal cord. Gently remove superficial layers of scar tissue by holding with fine forceps and cutting with fine scissors. With this method, re-open the site of laminectomy to expose the tissue containing the spinal cord injury area. Stop preparation when the dura suture is visually identified.</li>
		<li>Clamp the backbone by 2 stabilization clamps at spinous processes Th7 and Th10, and then elevate the animal to uncouple breathing movements from the vertebrae.</li>
		<li>With a small paperboard ruler measure the spinal cord area which is to be resected (length: 4 mm) and mark the edges of this respective tissue area with transverse incisions to allow the subsequent removal of tissue.</li>
		<li>Remove scar tissue via a combination of cutting and aspiration. Take out the tissue that has been separated from the spinal cord with the transverse incisions. Use gentle aspiration whenever it is sufficient to allow the removal of the tissue. In addition, whenever the stiff texture of the scar tissue makes tissue aspiration too difficult, use fine scissors to cut and remove this tissue.</li>
		<li>Insert a piece (approximately 5 mm x 5 mm x 5 mm cube) of hemostatic gelatin sponge into the tissue gap until bleeding subsides. The gelatin sponge will shrink in size as soon as it is soaked with liquid.</li>
	</ol>
	</li>
	<li>Implantation of PEG 600
	<ol>
		<li>Prepare 1 ml of pure undiluted PEG 600 for injection by heating to 37 &#176;C.</li>
		<li>Remove gelatin sponge.</li>
		<li>Insert sufficient amount (approximately 5 - 7 &#181;l) of PEG into the gap using a 10 &#181;l syringe or a 10 &#181;l pipette.</li>
		<li>Carefully cover the area with a piece (approximately 5 mm x 4 mm) of sealant/dura replacement.</li>
		<li>Fix the sealant to surrounding muscle tissue with a few drops of tissue glue. Place the sealant on top of the PEG-filled resection gap. To prevent slippage of the sealant, use small drops of tissue glue to fix the corners of the sealant to the surrounding muscle tissue. Note: Avoid leakage of the tissue glue onto the spinal cord tissue!</li>
	</ol>
	</li>
	<li>Closing of Tissue and Postoperative Care
	<ol>
		<li>Suture muscles and skin layer for layer with interrupted sutures with braided adsorbable 4-0 threads</li>
		<li>Inject 2 &#215; 2.5 ml of sodium chloride (NaCl [0.9%]) at 36 &#176;C s.c. for rehydration after the surgery. A single injection of 5 ml NaCl would stretch the animal&#8217;s skin and might cause unnecessary harm to the animal. Do not leave animal unattended until full consciousness is regained.</li>
		<li>Inject daily carprofen (s.c. 5 mg/kg) for at least 2 days after the surgery. House animal in single cage during the first 2 days, afterwards in groups of 2 - 3.</li>
		<li>Apply antibiotic treatment (daily oral administration of enrofloxacin) for the first post-operative week.</li>
		<li>Do manual bladder voiding at two to three times per day by gently stroking over the belly of the animal from rostral to caudal. Be careful not to move the vertebral column of the animal and don&#39;t lift the animal at the tail.&#160;Manually void the completely spinalized animal&#8217;s bladder daily during the whole survival time. Care must be taken to place food pellets and water bottles in a height which can be reached by the animals without standing on their hindpaws. Although being impaired in hind quarter function, the animals move and explore the cages actively immediately after surgery. It is recommended to keep the rats in sociable groups.</li>
		<li>For chronic lesions leave a survival time of the animals of five weeks before scar resection. Follow steps 1.4.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Ramer, L. M., Ramer, M. S., Bradbury, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoring+function+after+spinal+cord+injury:+towards+clinical+translation+of+experimental+strategies.">Restoring function after spinal cord injury: towards clinical translation of experimental strategies.</a> The Lancet. Neurology. 13 (12), 1241-1256 (2014).</li><li>McDonald, J. W., Howard, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repairing+the+damaged+spinal+cord:+a+summary+of+our+early+success+with+embryonic+stem+cell+transplantation+and+remyelination.">Repairing the damaged spinal cord: a summary of our early success with embryonic stem cell transplantation and remyelination.</a> Prog. Brain Res. 137, 299-309 (2002).</li><li>Yoon, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+spinal+cord+injury+treatment+using+autologous+bone+marrow+cell+transplantation+and+bone+marrow+stimulation+with+granulocyte+macrophage-colony+stimulating+factor:+Phase+I/II+clinical+trial.">Complete spinal cord injury treatment using autologous bone marrow cell transplantation and bone marrow stimulation with granulocyte macrophage-colony stimulating factor: Phase I/II clinical trial.</a> Stem Cells. 25 (8), 2066-2073 (2007).</li><li>Brotchi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+spinal+cord+tumor+resection.">Intrinsic spinal cord tumor resection.</a> Neurosurgery. 50 (5), 1059-1063 (2002).</li><li>Estrada, V., Tekinay, A., Muller, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+ECM+mimetics.">Neural ECM mimetics.</a> Prog. Brain Res. 214, 391-413 (2014).</li><li>Tetzlaff, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Systematic+Review+of+Cellular+Transplantation+Therapies+for+Spinal+Cord+Injury.">A Systematic Review of Cellular Transplantation Therapies for Spinal Cord Injury.</a> J.Neurotrauma. 28 (8), 1611-1682 (2010).</li><li>Brazda, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanical+microconnector+system+for+restoration+of+tissue+continuity+and+long-term+drug+application+into+the+injured+spinal+cord.">A mechanical microconnector system for restoration of tissue continuity and long-term drug application into the injured spinal cord.</a> Biomaterials. 34 (38), 10056-10064 (2013).</li><li>Estrada, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-lasting+significant+functional+improvement+in+chronic+severe+spinal+cord+injury+following+scar+resection+and+polyethylene+glycol+implantation.">Long-lasting significant functional improvement in chronic severe spinal cord injury following scar resection and polyethylene glycol implantation.</a> Neurobiol. Dis. 67, 165-179 (2014).</li><li>Rasouli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resection+of+glial+scar+following+spinal+cord+injury.">Resection of glial scar following spinal cord injury.</a> J.Orthop.Res. 27 (7), 931-936 (2009).</li><li>Schira, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significant+clinical,+neuropathological+and+behavioural+recovery+from+acute+spinal+cord+trauma+by+transplantation+of+a+well-defined+somatic+stem+cell+from+human+umbilical+cord+blood.">Significant clinical, neuropathological and behavioural recovery from acute spinal cord trauma by transplantation of a well-defined somatic stem cell from human umbilical cord blood.</a> Brain. 135, 431-446 (2011).</li></ol>

The authors have nothing to disclose.

Here two different surgical approaches are presented to bridge tissue gaps in the spinal cord after (1) acute complete transection and mMS implantation and (2) chronic spinal cord lesion and fibrous scar removal plus PEG matrix implantation. Both strategies lead to tissue preservation and axonal regeneration as well as to significant locomotor functional improvement of the treated animals. For mMS implantation an adequate fixation of the mMS within the spinal cord by the firm dura suture after surgery is a critical techn...

A traumatic injury to the spinal cord not only leads to the loss of axons but it further results in tissue defects which hinder any regenerative responses (for review see 1,2). Spinal cord tissue is often lost through secondary degeneration leading to cyst formation or holes in and around the lesion area. Most experimental therapeutic interventions focus on incomplete spinal cord damages like partial transection, crush or contusion injuries with a remaining rim of healthy tissue. For complete injuries like total transections resulting from traumatic accidents or surgical interventions, like tumor resections, only very limited treatment options are available today3,4. After complete transection, mechanic tension of the tissue results in spinal stump retraction, leaving a small gap in the spinal cord. Most strategies focus on filling this gap with tissue, cells or matrices5,6.
Here, a different strategy is presented, namely re-adaptation of the separated stumps using a novel microconnector device7. In order to readapt the two stumps, mechanical force has to be applied as a slight negative pressure to accomplish this (Figure 1). The mechanical microconnector system (mMS) is a multi-channel system of polymethylmethacrylate (PMMA) with honeycomb-shaped holes (Figure 1A) and provided with an outlet tubing system. It is implanted into the tissue gap resulting from complete spinal cord transection in the rat (Figure 1C). One tube can be connected to a vacuum pump to apply negative pressure to the mMS (Figure 1D). The pressure pulls the disconnected spinal cord stumps into the honeycomb-shaped holes of the mMS, which have microstructured walls to hold the tissue in place when pressure is released (Figure 1B). The tubing can be left intact after surgery and attached to an osmotic minipump in order to infuse substances into the lesion core (Figure 1E-F).
Besides an acute transection of the spinal cord another type of complete lesion results from surgical removal of a spinal tumor or a solid chronic lesion scar leading to large tissue gaps of several millimeters, which cannot be overcome by the mMS so far. The majority of patients with spinal cord trauma suffer from chronic injuries. In these patients, a fully developed scar occupies the lesion core. The surgical removal of the lesion scar is a concept for treatment which is currently investigated after experimental SCI8,9. While the resection procedure itself can be performed without causing considerable additional damage, the resulting tissue gap needs to be bridged with a suitable matrix which allows and promotes the regeneration of tissue and, in the specific case of spinal cord injuries, the regeneration of nerve fibers to maintain and promote locomotor functions. It was found that low-molecular weight polyethylene glycol (PEG 600) is a very suitable material for this purpose. Its lack of immunogenicity and the very low viscosity allow smooth integration into the surrounding tissue. Insertion of the biopolymer alone promotes the invasion of beneficial cells, including endothelial cells, peripheral Schwann cells, and astrocytes, and - very importantly - the regeneration and elongation of axons of descending and ascending fiber tracts as well as their ensheathment by compact myelin8. These regenerative responses were found to be accompanied by long-lasting functional improvements. The combination of resection of scar tissue and subsequent implantation of PEG 600 presents a safe and simple, yet very efficient means of bridging substantial spinal cord tissue defects.

<table><tbody><tr><td>PEG 600 Ph Eur&amp;nbsp;</td><td>Merck/VWR&amp;nbsp;</td><td>8,170,041,000</td><td></td></tr><tr><td>Gelastypt gelatine sponge&amp;nbsp;&amp;nbsp;</td><td>sanofi Aventis</td><td>PZN-8789582</td><td></td></tr><tr><td>Nescofilm Sealant&amp;nbsp;</td><td>Roth</td><td>2569.1</td><td></td></tr><tr><td>Baytril</td><td>Bayer</td><td></td><td></td></tr><tr><td>Rimadyl (Carpofen)</td><td>Pfizer</td><td></td><td></td></tr><tr><td>Forene (Isoflurane)</td><td>Abbvie</td><td></td><td></td></tr><tr><td>Kodan (skin disinfectant)</td><td></td><td></td><td></td></tr><tr><td>Histoacryl (tissue glue)</td><td></td><td></td><td></td></tr><tr><td>Friedman-Pearson Rongeur, 1 mm cup, straight&amp;nbsp;</td><td>Fine Science Tools</td><td>16020-14</td><td></td></tr><tr><td>Two-in-one Micro Spatula - 12 cm&amp;nbsp;</td><td>Fine Science Tools</td><td>10091-12</td><td></td></tr><tr><td>Dumont #7 Forceps - Inox Medical&amp;nbsp;</td><td>Fine Science Tools</td><td>11273-20</td><td></td></tr><tr><td>Dumont #5/45 Forceps - Inox Medical&amp;nbsp;</td><td>Fine Science Tools</td><td>11253-25</td><td></td></tr><tr><td>Spinal cord hook&amp;nbsp;</td><td>Fine Science Tools</td><td>10162-12</td><td></td></tr><tr><td>Scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>14078-10</td><td></td></tr><tr><td>Clamp&amp;nbsp;</td><td>Aesculap</td><td>EA016R</td><td></td></tr><tr><td>Ethicon Vicryl 4-0</td><td></td><td></td><td></td></tr><tr><td>Bepanthen Augen- und Nasensalbe</td><td>Bayer</td><td></td><td></td></tr><tr><td>Anatomical forceps&amp;nbsp;</td><td>Fine Science Tools</td><td>11000-13</td><td></td></tr><tr><td>Self-retaining retractor&amp;nbsp;</td><td>Fine Science Tools</td><td>17008-07</td><td></td></tr><tr><td>Skin clamp&amp;nbsp;</td><td>Fine Science Tools</td><td>13008-12</td><td></td></tr><tr><td>Aluspray&amp;nbsp;</td><td>Selectavet</td><td></td><td></td></tr></tbody></table>

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.

Tissue Preservation, Axonal Regrowth and Functional Benefit of mMS Implantation after Acute Complete Transection of the Spinal Cord 
It is demonstrated that the acute implantation of the mMS stabilized the completely transected spinal cord stumps and decreased shrinkage of the tissue (Figure 2A versus B). As visualized by Trichrome staining in sagittal sections, the green connective tissue staining of the fibrotic scar in th...

Watch this Scientific Journal Video about Experimental Strategies to Bridge Large Tissue Gaps in the Injured Spinal Cord after Acute and Chronic Lesion at JoVE.com

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

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 ...

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Research

JoVE Journal

Neuroscience

8.9K Views.  Heinrich-Heine-University Medical Center. 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.

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

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 ...

Medicine

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 ...

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

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

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

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

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 ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

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 ...

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human Brown-S&#233;quard syndrome&#160;is a common type of incomplete SCI caused by a lesion to one half of the spinal cord which results in paralysis and loss of&#160;proprioception&#160;on the same (or ipsilesional) side as the injury, and loss of pain and temperature sensation on the opposite (or contralesional) side. Adequate methodologies for producing a spinal cord lateral hemisection (HX) and assessing neurological impairments are essential to establish a reliable animal model of Brown-S&#233;quard syndrome. Although lateral hemisection model plays a pivotal role in basic and translational research, standardized protocols for creating such a hemisection and assessing unilateralized function are lacking. The goal of this study is to describe step-by-step procedures to produce a rat spinal lateral HX at the 9th thoracic (T9) vertebral level. We, then, describe a combined behavior scale for HX (CBS-HX) that provides a simple and sensitive assessment of asymmetric neurological performance for unilateral SCI. The CBS-HX, ranging from 0 to 18, is composed of 4 individual assessments which include unilateral hindlimb stepping (UHS), coupling, contact placing, and grid walking. For CBS-HX, the ipsilateral and contralateral hindlimbs are assessed separately. We found that, after a T9 HX, the ipsilateral hindlimb showed impaired behavior function whereas the contralateral hindlimb showed substantial recovery. The CBS-HX effectively discriminated behavioral functions between ipsilateral and contralateral hindlimbs and detected temporal progression of recovery of the ipsilateral hindlimb. The CBS-HX components can be analyzed separately or in combination with other measures when needed. Although we only provided visual descriptions of the surgical procedures and behavioral assessments of a thoracic HX, the principle may be applied to other incomplete SCIs and at other levels of the injury.

Here we describe surgical procedures to produce a reliable spinal cord lateral hemisection (HX) at the 9th thoracic level in adult rats and neurobehavioral assessments designed for detecting asymmetric deficits after such a unilateral injury.

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human ...

Thoracic Spinal Cord Hemisection Surgery and Open-Field Locomotor Assessment in the Rat

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are valuable tools to understand the neural mechanisms involved in locomotor recovery after SCI and to design therapies for clinical populations. There are several experimental SCI models including contusion, compression, and transection injuries that are used in a wide variety of species. A hemisection involves the unilateral transection of the spinal cord and disrupts all ascending and descending tracts on one side only. Spinal hemisection produces a highly selective and reproducible injury in comparison to contusion or compression techniques that is useful for investigating neural plasticity in spared and damaged pathways associated with functional recovery. We present a detailed step-by-step protocol for performing a thoracic hemisection at the T8 vertebral level in the rat that results in an initial paralysis of the hindlimb on the side of the lesion with graded spontaneous recovery of locomotor function over several weeks. We also provide a locomotor scoring protocol to assess functional recovery in the open-field. The locomotor assessment provides a linear recovery profile and can be performed both early and repeatedly after injury in order to accurately screen animals for appropriate time points in which to conduct more specialized behavioral testing. The hemisection technique presented can be readily adapted to other transection models and species, and the locomotor assessment can be used in a variety of SCI and other injury models to score locomotor function.

The rat thoracic spinal hemisection is a valuable and reproducible model of unilateral spinal cord injury to investigate the neural mechanisms of locomotor recovery and treatment efficacy. This article includes a detailed step-by-step guide to perform the hemisection procedure and to assess locomotor performance in an open-field arena.

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are ...

A Minimally Invasive, Fast Spinal Cord Lateral Hemisection Technique for Modeling Open Spinal Cord Injuries in Rats

Open spinal cord injury techniques modeling laceration-like injuries are time-consuming and invasive because they involve laminectomy. This new technique eliminates laminectomy by removing two spinous processes and lifting, then tilting the caudal vertebral arch. The surgical area opens up without the need for laminectomy. Lateral hemisection is then performed with direct visible control under a microscope. The trauma is minimized, requiring only a small bone wound. 
This technique has several advantages: it is faster and, therefore, less of a burden for the animal, and the bone wound is smaller. Because the laminectomy is eliminated, there is less chance for unwanted injury to the spinal cord, and there are no bone splinters that can cause problems (bone splinters embedded in the spinal cord can cause swelling and secondary damage). The vertebral canal remains intact. The main limitation is that the hemisection can only be performed in the intervertebral spaces. 
The results show that this technique can be performed much faster than the traditional surgical approach, using laminectomy (11 min vs. 35 min). This technique can be useful for researchers working with animal models of open spinal cord injury as it is widely adaptable and does not require any additional specialized instrumentation.

Here, we describe a new, fast technique modeling open spinal cord injury in rats that eliminates laminectomy. Lateral hemisection is performed while viewing through a microscope. The technique is versatile and can also be used in the cervical, thoracic, and lumbar regions of the spinal cord of other animals.

Open spinal cord injury techniques modeling laceration-like injuries are time-consuming and invasive because they involve laminectomy. This new technique ...