This work was supported by grants from the Christopher and Dana Reeve Foundation, the Medical Research Council, the European Research Council ECMneuro, and the Cambridge NHMRC Biomedical Research Center. We would like to express our deepest gratitude to Heleen Merel van &#8217;t Spijker and Justyna Barratt for their technical assistance during the filming. We would like to thank Dr. Elizabeth Moloney and Professor Joost Verhaagen (Netherlands Institute for Neuroscience) for assisting in AAV production.

All the following animal procedures were conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. If unfamiliar with these procedures, please check with local/national regulations and seek veterinary advice before starting the protocol.
1. Choosing a Suitable Strain of Animals
NOTE: A dorsal root crush injury results in the loss of sensation and paw deafferentation. Common adverse effects of forepaw deafferentation can include over-grooming, self-mutilatio...

<ol>
	<li>Chew, D. J., Fawcett, J. W., Andrews, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+challenges+of+long-distance+axon+regeneration+in+the+injured+CNS.">The challenges of long-distance axon regeneration in the injured CNS.</a> Prog Brain Res. 201, 253-294 (2012).</li><li>Wu, A., Lauschke, J. L., Morris, R., Waite, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+rat+forepaw+function+in+two+models+of+cervical+dorsal+root+injury.">Characterization of rat forepaw function in two models of cervical dorsal root injury.</a> J Neurotrauma. 26 (1), 17-29 (2009).</li><li>Hocquemiller, M., Giersch, L., Audrain, M., Parker, S., Cartier, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-Associated+Virus-Based+Gene+Therapy+for+CNS+Diseases.">Adeno-Associated Virus-Based Gene Therapy for CNS Diseases.</a> Hum Gene Ther. 27 (7), 478-496 (2016).</li><li>Cheah, M., 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=Expression+of+an+Activated+Integrin+Promotes+Long-Distance+Sensory+Axon+Regeneration+in+the+Spinal+Cord.">Expression of an Activated Integrin Promotes Long-Distance Sensory Axon Regeneration in the Spinal Cord.</a> J Neurosci. 36 (27), 7283-7297 (2016).</li><li>Andrews, M. R., 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=Alpha9+integrin+promotes+neurite+outgrowth+on+tenascin-C+and+enhances+sensory+axon+regeneration.">Alpha9 integrin promotes neurite outgrowth on tenascin-C and enhances sensory axon regeneration.</a> J Neurosci. 29 (17), 5546-5557 (2009).</li><li>Tan, C. L., 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=Kindlin-1+enhances+axon+growth+on+inhibitory+chondroitin+sulfate+proteoglycans+and+promotes+sensory+axon+regeneration.">Kindlin-1 enhances axon growth on inhibitory chondroitin sulfate proteoglycans and promotes sensory axon regeneration.</a> J Neurosci. 32 (21), 7325-7335 (2012).</li><li>McCarty, D. M., Young, S. M., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+adeno-associated+virus+(AAV)+and+recombinant+AAV+vectors.">Integration of adeno-associated virus (AAV) and recombinant AAV vectors.</a> Annu Rev Genet. 38, 819-845 (2004).</li><li>Mason, M. R., 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=Comparison+of+AAV+serotypes+for+gene+delivery+to+dorsal+root+ganglion+neurons.">Comparison of AAV serotypes for gene delivery to dorsal root ganglion neurons.</a> Mol Ther. 18 (4), 715-724 (2010).</li><li>Hermens, W. T., 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=Purification+of+recombinant+adeno-associated+virus+by+iodixanol+gradient+ultracentrifugation+allows+rapid+and+reproducible+preparation+of+vector+stocks+for+gene+transfer+in+the+nervous+system.">Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system.</a> Hum Gene Ther. 10 (11), 1885-1891 (1999).</li><li>Kappagantula, S., 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=Neu3+sialidase-mediated+ganglioside+conversion+is+necessary+for+axon+regeneration+and+is+blocked+in+CNS+axons.">Neu3 sialidase-mediated ganglioside conversion is necessary for axon regeneration and is blocked in CNS axons.</a> J Neurosci. 34 (7), 2477-2492 (2014).</li><li>Alto, L. T., 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=Chemotropic+guidance+facilitates+axonal+regeneration+and+synapse+formation+after+spinal+cord+injury.">Chemotropic guidance facilitates axonal regeneration and synapse formation after spinal cord injury.</a> Nat Neurosci. 12 (9), 1106-1113 (2009).</li><li>Bonner, J. F., 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=Grafted+neural+progenitors+integrate+and+restore+synaptic+connectivity+across+the+injured+spinal+cord.">Grafted neural progenitors integrate and restore synaptic connectivity across the injured spinal cord.</a> J Neurosci. 31 (12), 4675-4686 (2011).</li><li>Wang, R., 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=Persistent+restoration+of+sensory+function+by+immediate+or+delayed+systemic+artemin+after+dorsal+root+injury.">Persistent restoration of sensory function by immediate or delayed systemic artemin after dorsal root injury.</a> Nat Neurosci. 11 (4), 488-496 (2008).</li><li>Wong, L. E., Gibson, M. E., Arnold, H. M., Pepinsky, B., Frank, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artemin+promotes+functional+long-distance+axonal+regeneration+to+the+brainstem+after+dorsal+root+crush.">Artemin promotes functional long-distance axonal regeneration to the brainstem after dorsal root crush.</a> Proc Natl Acad Sci U S A. 112 (19), 6170-6175 (2015).</li><li>Tanguy, Y., 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=Systemic+AAVrh10+provides+higher+transgene+expression+than+AAV9+in+the+brain+and+the+spinal+cord+of+neonatal+mice.">Systemic AAVrh10 provides higher transgene expression than AAV9 in the brain and the spinal cord of neonatal mice.</a> Front Mol Neurosci. 8, 36 (2015).</li><li>Foust, K. D., Poirier, A., Pacak, C. A., Mandel, R. J., Flotte, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+intraperitoneal+or+intravenous+injections+of+recombinant+adeno-associated+virus+type+8+transduce+dorsal+root+ganglia+and+lower+motor+neurons.">Neonatal intraperitoneal or intravenous injections of recombinant adeno-associated virus type 8 transduce dorsal root ganglia and lower motor neurons.</a> Hum Gene Ther. 19 (1), 61-70 (2008).</li><li>Vulchanova, L., 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=Differential+adeno-associated+virus+mediated+gene+transfer+to+sensory+neurons+following+intrathecal+delivery+by+direct+lumbar+puncture.">Differential adeno-associated virus mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture.</a> Mol Pain. 6, 31 (2010).</li><li>Gray, S. 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=Directed+evolution+of+a+novel+adeno-associated+virus+(AAV)+vector+that+crosses+the+seizure-compromised+blood-brain+barrier+(BBB).">Directed evolution of a novel adeno-associated virus (AAV) vector that crosses the seizure-compromised blood-brain barrier (BBB).</a> Mol Ther. 18 (3), 570-578 (2010).</li><li>Fagoe, N. D., Attwell, C. L., Kouwenhoven, D., Verhaagen, J., Mason, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+ATF3+or+the+combination+of+ATF3,+c-Jun,+STAT3+and+Smad1+promotes+regeneration+of+the+central+axon+branch+of+sensory+neurons+but+without+synergistic+effects.">Overexpression of ATF3 or the combination of ATF3, c-Jun, STAT3 and Smad1 promotes regeneration of the central axon branch of sensory neurons but without synergistic effects.</a> Hum Mol Genet. 24 (23), 6788-6800 (2015).</li></ol>

James Fawcett is a paid consultant for Acorda Therapeutics and Novartis.

In this article, we present a step-by-step guide to perform a DRG injection and dorsal root crush injury in the lower cervical spinal cord of an adult rat. As this is an extremely invasive and delicate surgery, we strongly recommend that all potential users obtain sufficient training and practice before advancing to live animal surgery. The users should be familiar not only with spinal cord anatomy, but also with the surrounding muscle tissues, vertebral bone structure, and vasculature. Ideally, a competent user should b...

Achieving axon regeneration after nervous system injury is a challenging task1. To study the failure of axon regeneration in the central nervous system (CNS), researchers have used a plethora of nerve injury models. As regions of the CNS differ, it is important to use an anatomically appropriate model to study axon regeneration. By using the appropriate model, researchers can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration, as opposed to a &#34;one-for-all&#34; treatment strategy.
In spinal cord injury, for example, the most debilitating symptoms stem from the loss of sensation and locomotion. Loss of sensation is caused by damage to the ascending sensory pathways, while the loss of locomotion is caused by damage to the descending motor pathways. Due to cellular and anatomical differences between these two pathways, many targeted axon regeneration studies only focus on one or the other pathway, with the rationale that successful recovery of either would be of tremendous benefit to patients. In this article, we present a protocol that uses a direct dorsal root ganglia (DRG) injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration.
DRG sensory neurons are responsible for relaying sensory information, such as tactile sensation and pain, from the periphery to the CNS. The long axonal projections of sensory neurons in the spinal cord serve as a good model to study long-distance axon regeneration. In addition, as rodents can survive a sensory pathway lesion such as a dorsal root crush injury with minimal welfare complications, researchers can study CNS axon regeneration without the need to completely lesion the spinal cord. A quadruple C5 - C8 (cervical level 5 - 8) dorsal root crush injury has been shown to be a useful model for forepaw deafferentation2. Additionally, a dorsal root crush injury provides a &#34;cleaner&#34; model to study axon regeneration than a direct spinal cord injury because it is uncomplicated by other factors such as glial scar formation.
The use of viral gene therapy to reprogram neurons into a regenerative state has been increasingly regarded as a promising treatment strategy for many neurological conditions3. Studies have shown the application of an adeno-associated virus (AAV) vector carrying the transgene of a growth-promoting protein can achieve robust axon regeneration with behavioral recovery4,5,6. The apparent low pathogenicity of AAV in eliciting an immune response and the ability to transduce non-dividing cells, such as neurons, make it the optimal vector for gene therapy. Additionally, the recombinant AAV form is used for therapy. In this form, it is incapable of integrating its viral genome into the host genome7, reducing the risk of insertional mutagenesis as compared to other viral vectors, such as lentivirus. This makes AAV a safe choice for gene therapy applications.
As a DRG contains the cell bodies of sensory neurons, it is the most appropriate anatomical target for the administration of virus for gene therapy to study and/or promote sensory axon regeneration. In a study comparing different AAV serotypes and lentivirus, AAV serotype 5 (AAV5) was shown to be the most efficient in transducing DRG neurons over a time course of at least 12 weeks when injected directly into the DRG8. Additionally, AAV can achieve more than 40% transduction efficiency, transducing all DRG neuronal subtypes, such as the large-diameter neurofilament 200 kDa (NF200)-positive neurons and the small-diameter calcitonin gene-related peptide (CGRP)- or isolectin b4 (IB4)-positive neurons4,8.
As the surgical procedure of DRG injection and dorsal root crush injury is extremely invasive and delicate, we believe that this article will help new users to learn the procedure in a very efficient manner. In this article, we show representative results from adult rats four weeks after the injection of a control virus AAV5-GFP (green fluorescent protein) into C6 - C7 DRGs with a concurrent C5 - C8 dorsal root crush injury. This model is especially suitable for researchers who are investigating the use of viral gene therapy to promote sensory axon regeneration.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fast Green FCF dye</td>
			<td>Sigma-Aldrich</td>
			<td>F7258</td>
			<td>For visualizing colorless solution. Recommended concentration: 1%</td>
		</tr>
		<tr>
			<td>Cholera Toxin B subunit</td>
			<td>List Biological Laboratories</td>
			<td>104</td>
			<td>For anterograde axonal tracing. Recommended concentration: 1%</td>
		</tr>
		<tr>
			<td>IsoFlo</td>
			<td>Zoetis</td>
			<td>115095</td>
			<td>Inhalation anesthetic (active ingredient: isoflurane)</td>
		</tr>
		<tr>
			<td>Baytril 2.5% injectable</td>
			<td>Bayer</td>
			<td>05032756093017</td>
			<td>Antibiotic (active ingredient: enrofloxacin). Manufacturer&#39;s recommended dosage: 10 mg/kg</td>
		</tr>
		<tr>
			<td>Carprieve 5.0% w/v</td>
			<td>Norbrook</td>
			<td>02000/4229</td>
			<td>Analgesic (active ingredient: carprofen). Manufacturer&#39;s recommended dosage: 4 mg/kg</td>
		</tr>
		<tr>
			<td>Lacri-Lube</td>
			<td>Allergan</td>
			<td>PL 00426/0041</td>
			<td>Eye ointment</td>
		</tr>
		<tr>
			<td>Olsen-Hegar Needle Holder</td>
			<td>Fine Science Tools</td>
			<td>FST 12502-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Friedman Pearson Rongeur Curved 0.7mm Cup</td>
			<td>Fine Science Tools</td>
			<td>FST 16121-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Bonn Micro Forceps</td>
			<td>Fine Science Tools</td>
			<td>FST 11083-07</td>
			<td>For performing dorsal root crush injury</td>
		</tr>
		<tr>
			<td>Tissue Separating Scissors</td>
			<td>Fine Science Tools</td>
			<td>FST 14072-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>FST 14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Adson Forceps</td>
			<td>Fine Science Tools</td>
			<td>FST 11018-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Goldstein Retractor</td>
			<td>Fine Science Tools</td>
			<td>FST 17003-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors (straight)</td>
			<td>Fine Science Tools</td>
			<td>FST 15018-10</td>
			<td></td>
		</tr>
		<tr>
			<td>SURGIFOAM Absorbable Gelatin Sponge</td>
			<td>Ethicon</td>
			<td>1972</td>
			<td>For bleeding control</td>
		</tr>
		<tr>
			<td>Microliter Syringe RN701 (10 &#956;l)</td>
			<td>Hamilton</td>
			<td>80330</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom-made Removable Needle (for DRG injection)</td>
			<td>Hamilton</td>
			<td>7803-05</td>
			<td>33 gauge, 38 mm, point style 3</td>
		</tr>
		<tr>
			<td>Custom-made Removable Needle (for CTB injection)</td>
			<td>Hamilton</td>
			<td>7803-05</td>
			<td>33 gauge, 10 mm, point style 3</td>
		</tr>
		<tr>
			<td>UltraMicroPump with SYS-Micro4 Controller</td>
			<td>World Precision Instruments</td>
			<td>UMP3-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

dorsal root ganglion injection and dorsal root crush injury as a model for sensory axon regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

As a representation, a transverse spinal cord section with the attached DRG is presented to show the effectiveness of this protocol in transducing DRG neurons and tracing sensory axons in the spinal cord four weeks after injecting a control virus, AAV5-GFP, directly into the C7 DRG without a dorsal root crush injury (Figure 1A). Axons in both the dorsal column and the dorsal horn of the spinal cord express GFP (Figure 1B), as well as the cell bodies and a...

Watch this Scientific Journal Video about Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration at JoVE.com

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

The goal of this surgical approach is to directly apply treatments at the level of the neuronal cell body, and assess axon regeneration following therapeutic intervention, without having to perform a more damaging lesion within the central nervous system. This method can help answer key questions in the field of axon regeneration, such as, can therapeutically treated axons regenerate in the spinal cord, and possibly reach the brain stem targets? The main advantage of this technique is that researchers can examine CNS axon regeneration without having to directly injure the spinal cord.This reduces post-surgical complication, and improves animal welfare. Begin by properly anesthetizing the animal. Confirming a surgical plane of anesthesia by the absence of a reaction to a toe pinch.Then weigh the animal and record the preoperative weight. For a surgical DRG injection, shave the fur on the neck from between the ears to just below to the scapuli. Disinfect the shaved area with an antiseptic product.Inject two milliliters of saline subcutaneously on the flank of the animal, along with an appropriate dose of analgesic and antibiotic. Apply eye ointment to both eyes to prevent corneal drying during the procedure. Place the animal in a spinal stereotaxic frame, and place a heating pad at 37 degrees Celsius underneath the animal.Begin the surgery by locating the prominent C2 and T2 spinous processes over the skin. Then use a number 10 scalpel to make a skin incision between the C2 and T2 spinous processes. Once the skin is opened, a white fibrous tissue midline should be visible on the first layer of muscle, which has a jellylike texture.Make a similar sized incision on the first layer of muscle along the white midline. Do not go beyond the first prominent T2 spinous process, as there is a major blood vessel located near there. Retract the first layer of muscle using two retractors, one placed rostrally, and one caudally.The second layer of muscle with a striated appearance should now be visible. Locate the midline of the second layer of muscle, where two longitudinal muscles can be observed, connected by a thin, membranous tissue. Then use a pair of micro scissors to dissect the membranous tissue and separate the two longitudinal muscles.Adjust the retractors to expose the third layer of thin muscle covering the spine. The spinous processes can be felt by lightly touching with a pair of forceps over the third layer of muscle. Use micro scissors to make a small incision on the third layer of muscle, and gently scrape off the muscle from the bone, using a curette or scalpel in a sideways manner, to clearly expose the vertebrae.To expose the left C5 to C8 DRG's, use a pair of fine bone shears to perform left hemi-laminectomy on the C4 to T1 vertebrae, by carefully removing part of the lamina and pedicle. Once enough of the DRG has been exposed for injection, prepare the syringe by placing the virus-filled microliter syringe fitted with a custom-made 33 gauge blunt needle onto the stereotaxic syringe holder. Next, use a 30 gauge beveled needle to make a small, superficial opening on each of the targeted DRG to assist with the insertion of the injection needle.Gently adjust the stereotaxic coordinates to insert the 33 gauge needle into the center of the DRG. Ideally, the needle should be placed at the very center of the DRG to ensure uniform diffusion of the injector solution throughout the DRG. Once the needle is inserted, use the infusion syringe pump to inject one microliter of the virus into each DRG.During the injection, the DRG will slowly change color if the virus solution contains a colored dye. Do not over-insert the needle, as this may cause fluid to leak out from the ventral side of the DRG. Should leakage occur during injection, adjust the position of the needle immediately.Three minutes after the end of the injection, withdraw the injection needle. To perform a concurrent C5 to C8 dorsal root crush injury, crush each root three times for 10 seconds, using a pair of fine-tipped forceps. Completely oppose the ends of the forceps.A white line in the tissue should appear at the crush site. Following the surgeries, ensure that there is no bleeding or small pieces of bone fragments left at the incision site before closing up. If preferred, place a small piece of surgical absorbable sponge on top of the exposed spinal cord and DRG.Allow the third layer of the muscle to retract back naturally onto the spine, without suturing. Loosely suture the two longitudinal muscles on the second layer with absorbable 6-0 suture material. Suture the first layer of muscle with absorbable 6-0 suture material.Suture the skin with absorbable 5-0 suture material. If heavy bleeding occurred during the surgery, subcutaneously inject one to two milliliters of saline to replenish the loss of fluid from the animal, as permitted under local regulations. Provide edible hydrating gel, and allow the animal to recover fully from anesthesia.Frequently monitor the animal for at least one hour before returning to the holding area. The following images show recovery of post-surgical animals at day one, day seven, after skin suture removal, and at day 14. One week prior to tissue collection, properly anesthetize the animal as before, and then stabilize the left forepaw by taping the limb to the table.Before injection of CTB, use a 30 gauge beveled needle to make a small, superficial opening on the skin to assist with the insertion of the injection needle. Then use a microliter syringe fitted with a custom-made 33 gauge short needle, to slowly inject one microliter of 1%CTB subcutaneously into the center of the glabrous footpad and four digits. Allow the animal to recover fully from anesthesia, before returning to the holding area.This image shows the result of the DRG injection without dorsal root crush injury. GFP positive cell bodies and axons in the spinal cord are clearly visible four weeks after the injection of AAV5GFP. This higher magnification image shows the axons in the dorsal column and dorsal horn.The GFP labeled cell bodies in the DRG are seen here. This image shows CTB positive axon terminals in the Cuneate nucleus one week following CTB injection. The following images show results of a complete dorsal root crush injury.As seen here, GFP labeled axotomized sensory axons can regenerate up to the dorsal root entry zone, but not into the spinal cord after injury. This is an image of the Cuneate nucleus in the same animal. CTB positive axon terminals are absent following a complete dorsal root crush injury.The presence of labeled axons in the spinal cord after injury represents either incomplete injury or regeneration. The presence of CTB positive terminals in the Cuneate nucleus suggests incomplete injury, while their absence suggest complete injury and potentially partial regeneration into the spinal cord. While attending this procedure, it is important to remove just the right amount of vertebrae bones to expose the DRG and dorsal root, without inducing any unwanted damage, especially to the spinal cord.Otherwise, this may result in unexpected post-surgical complications. After watching this video, you should have a good understanding of how to perform a DRG injection. You can incorporate in other techniques such as central spinal injury, electrophysiology, behavior testing, and anatomical analysis to examine axon regenerations.

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Research

JoVE Journal

Neuroscience

17.2K Görüntüleme.  University of Cambridge. The goal of this surgical approach is to directly apply treatments at the level of the neuronal cell body, and assess axon regeneration following therapeutic intervention, without having to perform a more damaging lesion within the central nervous system. This method can help answer key questions in the field of axon regeneration, such as, can therapeutically treated axons regenerate in the spinal cord, and possibly reach the brain stem targets? The main advantage of this technique is that re...