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>
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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.

Research

JoVE Journal

Neuroscience

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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 ...

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) ...