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 border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PEG 600 Ph Eur&#160;</td>
			<td style="width:71px;">Merck/VWR&#160;</td>
			<td align="right" style="width:119px;">8,170,041,000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelastypt gelatine sponge&#160;&#160;</td>
			<td>sanofi Aventis</td>
			<td>PZN-8789582</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nescofilm Sealant&#160;</td>
			<td>Roth</td>
			<td>2569.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Baytril</td>
			<td>Bayer</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rimadyl (Carpofen)</td>
			<td>Pfizer</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forene (Isoflurane)</td>
			<td>Abbvie</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kodan (skin disinfectant)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histoacryl (tissue glue)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Friedman-Pearson Rongeur, 1 mm cup, straight&#160;</td>
			<td>Fine Science Tools</td>
			<td>16020-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Two-in-one Micro Spatula - 12 cm&#160;</td>
			<td>Fine Science Tools</td>
			<td>10091-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #7 Forceps - Inox Medical&#160;</td>
			<td>Fine Science Tools</td>
			<td>11273-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5/45 Forceps - Inox Medical&#160;</td>
			<td>Fine Science Tools</td>
			<td>11253-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spinal cord hook&#160;</td>
			<td>Fine Science Tools</td>
			<td>10162-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissors&#160;</td>
			<td>Fine Science Tools</td>
			<td>14078-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clamp&#160;</td>
			<td>Aesculap</td>
			<td>EA016R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethicon Vicryl 4-0</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bepanthen Augen- und Nasensalbe</td>
			<td>Bayer</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anatomical forceps&#160;</td>
			<td>Fine Science Tools</td>
			<td>11000-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Self-retaining retractor&#160;</td>
			<td>Fine Science Tools</td>
			<td>17008-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Skin clamp&#160;</td>
			<td>Fine Science Tools</td>
			<td>13008-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluspray&#160;</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 ...

experimental-strategies-to-bridge-large-tissue-gaps-injured-spinal

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Diffusion Imaging in the Rat Cervical Spinal Cord

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a diagnostic and prognostic tool in cases of disease or injury. Diffusion weighted imaging (DWI), is sensitive to the thermal motion of water molecules and allows for inferences of tissue microstructure. This report describes a protocol to acquire and analyze DWI of the rat cervical spinal cord on a small-bore animal system. It demonstrates an imaging setup for the live anesthetized animal and recommends a DWI acquisition protocol for high-quality imaging, which includes stabilization of the cord and control of respiratory motion. Measurements with diffusion weighting along different directions and magnitudes (b-values) are used. Finally, several mathematical models of the resulting signal are used to derive maps of the diffusion processes within the spinal cord tissue that provide insight into the normal cord and can be used to monitor injury or disease processes noninvasively.

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost oligodendrocytes, myelin, axons, and neurons. Remyelination is an endogenous repair mechanism whereby new myelin is produced subsequent to proliferation, recruitment, and differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes, and is necessary to protect axons from further damage. Currently, all therapeutics for the treatment of multiple sclerosis target the aberrant immune component of the disease, which reduce inflammatory relapses but do not prevent progression to irreversible neurological decline. It is therefore imperative that remyelination-promoting strategies be developed which may delay disease progression and perhaps reverse neurological symptoms. Several animal models of demyelination exist, including experimental autoimmune encephalomyelitis and curprizone; however, there are limitations in their use for studying remyelination. A more robust approach is the focal injection of toxins into the central nervous system, including the detergent lysolecithin into the spinal cord white matter of rodents. In this protocol, we demonstrate that the surgical procedure involved in injecting lysolecithin into the ventral white matter of mice is fast, cost-effective, and requires no additional materials than those commercially available. This procedure is important not only for studying the normal events involved in the remyelination process, but also as a pre-clinical tool for screening candidate remyelination-promoting therapeutics.

Demyelinating diseases can be modeled in animals by focal application of lysolecithin into the CNS. A single injection of lysolecithin into mouse spinal cord produces a lesion that spontaneously repairs over time. The goal is to study factors involved in de- and remyelination, and to test agents for enhancing repair.

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...