Name: a minimally invasive, fast spinal cord lateral hemisection technique for modeling open spinal cord injuries in rats
Uploaded: 2022-03-23T13:00:00
Description: Watch this Scientific Journal Video about A Minimally Invasive, Fast Spinal Cord Lateral Hemisection Technique for Modeling Open Spinal Cord Injuries in Rats at JoVE.com

The authors wish to thank Gergely &#193;ngy&#225;n for the original artwork. This research work was funded by Semmelweis University, Budapest, Hungary. This study was also supported by the Hungarian Human Resources Development Operational Program (EFOP-3.6.2-16-2017-00006). Additional support was received from the Thematic Excellence Programme (2020-4.1.1.-TKP2020) of the Ministry for Innovation and Technology in Hungary, within the framework of the Therapy thematic program of Semmelweis University.

All animal procedures were carried out according to the EU Directive (2010/63/EU) and were approved by the animal ethics committee of the Hungarian National Food Chain Safety Office (PEI/001/2894-11/2014). All applicable institutional and governmental regulations concerning the ethical use of animals were followed during this study.
1. Preparation before surgery
<ol>
	<li>Sterilize all instruments used during the procedure (see the Table of Materials) and di...

<ol>
	<li>Failli, 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=Functional+neurological+recovery+after+spinal+cord+injury+is+impaired+in+patients+with+infections.">Functional neurological recovery after spinal cord injury is impaired in patients with infections.</a> Brain. 135, 3238-3250 (2012).</li><li>Guan, B., Chen, R., Zhong, M., Liu, N., Chen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effect+of+Oxymatrine+against+acute+spinal+cord+injury+in+rats+via+modulating+oxidative+stress,+inflammation+and+apoptosis.">Protective effect of Oxymatrine against acute spinal cord injury in rats via modulating oxidative stress, inflammation and apoptosis.</a> Metabolic Brain Disease. 35 (1), 149-157 (2020).</li><li>Kjell, J., Olson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+models+of+spinal+cord+injury:+from+pathology+to+potential+therapies.">Rat models of spinal cord injury: from pathology to potential therapies.</a> Disease Models &amp; Mechanisms. 9 (10), 1125-1137 (2016).</li><li>Minakov, A. N., Chernov, A. S., Asutin, D. S., Konovalov, N. A., Telegin, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+spinal+cord+injury+in+laboratory+rats.">Experimental models of spinal cord injury in laboratory rats.</a> Acta Naturae. 10 (3), 4-10 (2018).</li><li>Borb&#233;ly, Z., 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=Effect+of+rat+spinal+cord+injury+(hemisection)+on+the+ex+vivo+uptake+and+release+of+[3H]noradrenaline+from+a+slice+preparation.">Effect of rat spinal cord injury (hemisection) on the ex vivo uptake and release of [3H]noradrenaline from a slice preparation.</a> Brain Research Bulletin. 131, 150-155 (2017).</li><li>Brown, A. R., Martinez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thoracic+spinal+cord+hemisection+surgery+and+open-field+locomotor+assessment+in+the+rat.">Thoracic spinal cord hemisection surgery and open-field locomotor assessment in the rat.</a> Journal of Visualized Experiments: JoVE. (148), e59738 (2019).</li><li>Taoka, Y., Okajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+injury+in+the+rat.">Spinal cord injury in the rat.</a> Progress in Neurobiology. 56 (3), 341-358 (1998).</li><li>Hou, S., Saltos, T. M., Iredia, I. W., Tom, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+techniques+influence+local+environment+of+injured+spinal+cord+and+cause+various+grafted+cell+survival+and+integration.">Surgical techniques influence local environment of injured spinal cord and cause various grafted cell survival and integration.</a> Journal of Neuroscience Methods. 293, 144-150 (2018).</li><li>Mattucci, 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=Development+of+a+traumatic+cervical+dislocation+spinal+cord+injury+model+with+residual+compression+in+the+rat.">Development of a traumatic cervical dislocation spinal cord injury model with residual compression in the rat.</a> Journal of Neuroscience Methods. 322, 58-70 (2019).</li><li>Ahmed, R. U., Alam, M., Zheng, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+spinal+cord+injury+and+behavioral+tests+in+laboratory+rats.">Experimental spinal cord injury and behavioral tests in laboratory rats.</a> Heliyon. 5 (3), 01324 (2019).</li><li>Ashammakhi, 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=Regenerative+therapies+for+spinal+cord+injury.">Regenerative therapies for spinal cord injury.</a> Tissue Engineering. Part B, Reviews. 25 (6), 471-491 (2019).</li></ol>

This minimally invasive spinal cord injury technique was developed when studying spinal cord-injured rats, and the team was faced with problems arising from the surgery itself (bone splinters from the laminectomy causing compression and damaging the spinal cord, surgery taking too long, slow healing of a large bone wound). By eliminating the laminectomy, the procedure became much faster (11 min vs. 35 min), the structure of the vertebral canal remained intact, the bone wound was much smaller, and there were no bone splin...

Spinal cord injuries (SCIs) are unfortunately prevalent injuries in humans. SCIs can be complicated in different ways, for example, by infections, and it is clinically important to study these injuries1. Because there is no single, definite cure for SCIs, animal models are still needed to further the understanding of researchers and advance possible treatments2,3. Although closed injuries are most commonly modeled (compression and contusion), it is clinically important to understand lacerations, which can only be modeled in open injuries4. Open-wound models using transection or hemisection can be used to demonstrate a more precise localization of a wound compared to closed injury models, owing to the nature of the injury (contusion vs. surgical cut). Open-wound experiments can shed light on more specific neuronal injuries in a controlled, reliable, and replicable way5. The complete or partial transection of the spinal cord is a widely used open-wound technique and can be viewed in detail in the article by Brown and Martinez6.
When studying open spinal cord injury in rats, several animals presented problems that arose from the surgery: bone splinters from the laminectomy became embedded in the spinal cord and caused swelling; the larger bone wound needed a long time to heal; the surgery took too long. An alternate surgical technique was developed to eliminate these problems. The goal was to develop a faster technique that is gentler for the animal. This newly developed technique is much faster than traditional SCI techniques. The surgical approach is minimally invasive, resulting in a smaller bone wound while eliminating problems arising from the laminectomy.
All open-wound techniques involve opening the dura7. Several recent studies have examined different, newly developed techniques, aiming to improve the previous methods8,9. Even though the opening of the dura cannot be excluded using this new technique, it causes a smaller wound on the dura while offering a reliable, controlled injury of the spinal cord. Consulting the literature on spinal cord injury techniques, many authors tried to minimize the time of surgery by implementing minor changes to the original technique10. Laminectomy is always part of these surgical procedures, although it is time-consuming and requires a larger bone wound to be made6. This surgical technique can be appropriate for researchers using open wound spinal cord injury models, specifically complete transection or lateral hemisection performed in the intervertebral spaces (Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Augmentin (1,000 mg/200 mg powder)</td>
			<td>GlaxoSmithKline, UK</td>
			<td></td>
			<td>One-time dose of s.c. antibiotics prophylactically (10 mg of amoxicillin and 2 mg clavulanic acid; Augmentin 1,000 mg/200 mg powder). Every day following surgery, 10 mg of amoxicillin and 2 mg of clavulanic acid (Augmentin 1,000 mg/200 mg powder) per day per animal</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>EGIS, Hungary</td>
			<td></td>
			<td>Disinfect the skin of the surgical area using a povidone-iodine solution</td>
		</tr>
		<tr>
			<td>Calypsol (50 mg/mL)</td>
			<td>Richter Gedeon, Hungary</td>
			<td></td>
			<td>Anesthesia: combination of ketamine 80 mg/kg and xylazine 8 mg/kg intramuscularly</td>
		</tr>
		<tr>
			<td>CP XYLAZIN 2% (20 mg/mL)</td>
			<td>Produlab Pharma B.V., the Netherlands</td>
			<td></td>
			<td>Anesthesia: combination of ketamine 80 mg/kg and xylazine 8 mg/kg intramuscularly</td>
		</tr>
		<tr>
			<td>Dental bone forceps</td>
			<td>Dentech, Hungary</td>
			<td>BS 0127</td>
			<td>Remove the spinous processes of the 13th thoracic vertebra and the 1st lumbar vertebra using dental bone forceps</td>
		</tr>
		<tr>
			<td>dental surgical micromotor</td>
			<td>W&#38;H, Austria</td>
			<td>MF-TECTORQUE</td>
			<td>Using a dental surgical micromotor, a laminectomy is performed at the L1 vertebra</td>
		</tr>
		<tr>
			<td>optical microscope</td>
			<td>Zeiss, Germany</td>
			<td>OPMI19-FC</td>
			<td>Control the procedure by viewing an enlarged (16x magnification) microscopic image</td>
		</tr>
		<tr>
			<td>physiological saline solution (0.9% NaCl)</td>
			<td>Fresenius Kabi, Germany</td>
			<td></td>
			<td>Keep the rat&#39;s eyes moist throughout the entire anesthesia using physiological saline solution drops (reapply as necessary)</td>
		</tr>
		<tr>
			<td>raspatorium</td>
			<td>Dentech, Hungary</td>
			<td>FK 1164</td>
			<td>Dissect the muscles attached to the vertebrae with the aid of a raspatorium, until all the spinal ligaments are visible.</td>
		</tr>
		<tr>
			<td>retractor</td>
			<td>Dentech, Hungary</td>
			<td>RT 1253</td>
			<td></td>
		</tr>
		<tr>
			<td>scalpel</td>
			<td>Dentech, Hungary</td>
			<td>BB 173</td>
			<td></td>
		</tr>
		<tr>
			<td>scalpel</td>
			<td>Dentech, Hungary</td>
			<td>BB 184</td>
			<td></td>
		</tr>
		<tr>
			<td>scalpel blade 12</td>
			<td>B. Braun, Germany</td>
			<td>12</td>
			<td></td>
		</tr>
		<tr>
			<td>scalpel blade 20</td>
			<td>B. Braun, Germany</td>
			<td>20</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile cut gauze 10 x 10 cm</td>
			<td>Sterilux, Hartmann, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sutures (monofilament, synthetic; absorbable and nonabsorbable), size: 4-0</td>
			<td>B. Braun, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tweezer (13 cm)</td>
			<td>Dentech, Hungary</td>
			<td>BD 1555</td>
			<td></td>
		</tr>
		<tr>
			<td>tweezer (delicate tissue forceps)</td>
			<td>Dentech, Hungary</td>
			<td>BD 1670</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Following the hemisection, the rats show paralysis in the ipsilateral hindlimb (in vivo proof of successful hemisection). Thorough specimen evaluation can only be done following the removal of the spinal cord (see Figure 2, where the removed spinal cord can be seen from both the ventral and dorsal sides).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63534/63534fig02.jpg" /> 
Figure 2...

Watch this Scientific Journal Video about A Minimally Invasive, Fast Spinal Cord Lateral Hemisection Technique for Modeling Open Spinal Cord Injuries in Rats at JoVE.com

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

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 ...

This protocol is significant because spinal cord injuries are still prevalent today, and research is needed to further the understanding of these injuries and develop possible therapies. This technique is more accurate and reproducible than compression and contusion injuries and less invasive than injuries with complete laminectomy. So it might be a better option for a simulation of spinal cord injury than previously described procedures.To begin, use a scalpel blade 20 to open the surgical area by placing a two to 2.5 centimeter long incision through all the layers of the skin along the spine. Position this incision parallel to the spinal column using the L1 vertebra as a midpoint, extending it approximately one centimeter in both the cranial and coddle direction along the spinal column. Mobilize the sides of the wound by cutting through the connective tissue surrounding the muscles.Then place two parallel incisions along the spine, penetrating the periosteum. Place the incisions right next to the spinal processes on both sides, spanning the distance between the thoracic 13 and L1 vertebrae. Dissect the muscles attached to the vertebrae with the eight of a respirator until all the spinal ligaments are visible.Place a retractor. Then remove the spinal processes of the 13th thoracic vertebra and the first lumbar vertebra Using dental bone forceps to visualize the entire surgical area. Use sterile gauze to control bleeding throughout the procedure when necessary.Carefully lift the remainder of the L1 spinal processes, elevating the L1 vertebra arch. Remove the ligamentum flavum to access the spinal cord. Raise the coddle spinal processes further, allowing access to the severed spinal dura mater.Tip the coddle spinal processes in the cranial direction to visualize the pia mater. Look through the pia mater for the posterior median vein, showcasing the midline of the spine. Using the vein as a directional bisector, place an incision using a microsurgical scalpel while sparing the vein.Place the incision under the vein in the transversal plane through the anterior posterior diameter of the spinal cord. Perform the hemisection by moving the blade laterally away from the center line. Place the incision to avoid cutting the anterior spinal artery.Ensure excess pressure is not applied on the vertebral body when cutting the spinal cord to spare the anterior spinal artery on the ventral side of the spinal cord. Do not close the dura mater directly during wound closure. Tightly suture the muscles and spinal processes, indirectly closing the small wound on the dura mater.Close the dorsal connective tissue layer with sutures. This new minimally invasive spinal cord injury method is performed much faster than the traditional surgery approach using laminectomy and does not require additional specialized instrumentation. Hematoxylin and eosin staining were used to visualize the injury site.Other sections can be performed along the spinal cord mimicking specific neural injuries. Regeneration can also be studied since electrodes or stem cells can be placed using this method. Most techniques utilizing open wound models of spinal cord injury implementing either hemisection or transaction can be simplified and sped up using this technique.

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Research

JoVE Journal

Neuroscience

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 ...

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

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 ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

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 ...

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) ...

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 ...