Name: thoracic spinal cord hemisection surgery and open-field locomotor assessment in the rat
Uploaded: 2019-06-26T14:30:00
Description: Watch this Scientific Journal Video about Thoracic Spinal Cord Hemisection Surgery and Open-Field Locomotor Assessment in the Rat at JoVE.com

This work was supported by the Canadian Institutes for Health Research (CIHR; MOP-142288) to M.M. M.M. was supported by a salary award from Fonds de Recherche Qu&#233;bec Sant&#233; (FRQS), and A.R.B was supported by a fellowship from FRQS.

The experiments described in this article were performed in compliance with the guidelines of the Canadian Council on Animal Care and were approved by the ethics committee at the Universit&#233; de Montr&#233;al.
1. Thoracic hemisection surgery
<ol>
	<li>Wear appropriate protective equipment (gloves, mask, and gown) to maintain an aseptic environment for surgery. Clean the surgical area with alcohol wipes, and place sterile surgical drapes over the surgical field. Sterilize surgical tools an...

A major strength of the hemisection technique is the selectivity and reproducibility of the lesion which leads to reduced variability in histological and behavioral phenotypes between animals25. In order to ensure a unilateral lesion at the appropriate spinal level, accurate identification of both the proper vertebral segment and spinal cord midline is critical. As there can be a tendency for the spinal cord to rotate in the direction of the cut during the hemisection procedure, it can be benefici...

Spinal cord injury (SCI) is associated with severe disturbances in motor, sensory, and autonomic function. Experimental animal models of SCI are valuable tools to understand the anatomical and physiological events involved in SCI pathology, to investigate the neural mechanisms in repair and recovery, and to screen for efficacy and safety of potential therapeutic interventions. The rat is the most commonly used species in SCI research1. Rat models are low cost, easy to reproduce, and a large battery of behavioral tests are available to assess functional outcomes2. Despite some differences in tract locations, the rat spinal cord shares overall similar sensorimotor functions with larger mammals, including primates3,4. Rats also share analogous physiological and behavioral consequences to SCI that relate to humans5. Non-human primate and large animal models can provide a closer approximation of human SCI6 and are essential to prove treatment safety and efficacy prior to human experimentation, but are less commonly used due to ethical and animal welfare considerations, expenses, and regulatory requirements7.
Rat transection SCI models are performed by the targeted interruption of the spinal cord with a selective lesion using a dissection knife or iridectomy scissors after a laminectomy. Compared to a complete transection, partial transection in the rat results in a less severe injury, easier postoperative animal care, spontaneous locomotor recovery, and more closely models SCI in humans which is predominately incomplete with partial sparing of tissue connecting the spinal cord and supraspinal structures8. A unilateral hemisection disrupts all ascending and descending tracts on one side only, and produces quantifiable and highly reproducible locomotor deficits, enhancing exploration of the underlying biological mechanisms. The most prominent functional consequence of the hemisection is an initial limb paralysis on the same side and below the level of the lesion with graded spontaneous recovery of locomotor function over several weeks9,10,11,12. The hemisection model is particularly useful to investigate neural plasticity of damaged and residual tracts and circuits associated with functional recovery9,11,12,13,14,15,16,17,18. Specifically, hemisection performed at the thoracic level, i.e., above the spinal circuits that control hindlimb locomotion, is particularly useful for investigating changes in locomotor control. As a non-linear relationship exists between lesion severity and locomotor recovery after SCI19, appropriate behavioral testing to assess functional outcomes is paramount in experimental models.
A comprehensive battery of behavioral tests are available to assess specific aspects of functional locomotor recovery in the rat2,20. Many locomotor tests do not provide reliable measures early after SCI as rats are too disabled to support their body weight. A measure of spontaneous locomotor performance that is sensitive to deficits early after injury, and does not require preoperative training or specialized equipment, is beneficial in order to monitor locomotor recovery for appropriate time points in which to supplement specialized behavioral testing. The Martinez open-field assessment score10, originally developed for evaluating locomotor performance after cervical SCI in the rat, is a 20-point ordinal score assessing global locomotor performance during spontaneous overground locomotion in an open-field. Scoring is conducted separately for each limb using a rubric that evaluates specific parameters of a range of locomotor measures including articular limb movement, weight support, digit position, stepping abilities, forelimb-hindlimb coordination, and tail position. The assessment score is derived from the Basso, Beattie and Bresnahan (BBB) open-field rating scale designed to evaluate locomotor performance after thoracic contusion21. It is adapted to accurately and reliably evaluate both forelimb and hindlimb locomotor function, allows for independent assessment of the different scoring parameters that is not amenable with the hierarchical scoring of the BBB, and provides a linear recovery profile10. Additionally, in comparison to the BBB, the assessment score is sensitive and reliable in more severe injury models10,11,20,22. The assessment score has been used to assess locomotor impairment in the rat following cervical10,12 and thoracic9 SCI alone and in combination with traumatic brain injury23.
We present here a detailed step-by-step protocol for performing a thoracic hemisection SCI at the T8 vertebral level in the female Long-Evans rat, and for assessing hindlimb locomotor recovery in the open-field.

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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.

Reproducible lesions with a high degree of consistency can be generated with the hemisection technique. To assess and compare lesions sizes between experimental groups, the maximal area of the lesion as a percentage of the total cross-section of the spinal cord can be readily calculated with histological staining of spinal cord sections. Figure 1 shows a representative lesion of the left hemicord and an overlay of the proportion of maximal lesion area shared between rats with a mean lesion s...

Watch this Scientific Journal Video about Thoracic Spinal Cord Hemisection Surgery and Open-Field Locomotor Assessment in the Rat at JoVE.com

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

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 ...

Spinal hemisection produces quantifiable and ideal reproducible locomotor deficits that can be captured with the locomotive scale that we have developed. The main advantage of this technique is the selectivity and reproducibility of the lesion, which leads to a reduced variability in behavioral phenotypes between animals. After confirming a lack of response to toe pinch in an anesthetized adult rat, make a 2.5 centimeter incision in the shaved skin overlying the T-6 to T-10 vertebrae and use blunt scissors to retract the skin and superficial fat.Use blunt dissection scissors and a self-retaining retractor to separate the paravertebral muscles, inserting on the dorsal aspect of the T-7 to T-9 vertebrae and use fine forceps and cotton-tipped applicators to debride and clear any remaining tissue to expose the spinous processes and vertebral lamina. Place the rate under a stereo microscope and use delicate bone trimmers to carefully cut the facets bilaterally on the T-7 and T-8 vertebrae. Use a scalpel to make a one-millimeter superficial cut in the dorsal connective tissue between the T-8 and T-9 vertebral laminae, taking care not to injure the underlying cord and use bone trimmers to remove the spinous process of the T-8 vertebra.With curved hemostatic forceps carefully clamped on the T-7 spinous process, rotate the caudal end of the T-8 laminae slightly rostrally approximately 20 degrees and insert the bone trimmers under the T-8 lamina. Make a midline cut extending along the lamina, continuing the laminectomy by repeating the cuts on the left and right side of the vertebral lamina medial to the transverse processes to expose the spinal cord. Drip 100 microliters of 2%lidocaine into the exposed spinal canal and use fine forceps and iridectomy scissors to remove the dura overlaying the T-8 spinal segment.Repeat the lidocaine administration to the exposed cord and identify the midline of the cord by visualization of a center line created between the spinous processes extending between the exposed T-7 to T-9 vertebra. Using fine forceps to stabilize the spinal cord, use a dissecting knife to hemisect the spinal cord from the midline toward one side of the animal, taking care not to cut through the anterior spinal artery on the ventral side. Using iridectomy scissors, carefully cut through any remaining tissue on the lesion side of the spinal cord to ensure the ventral-lateral quadrant is appropriately transected and place a sterile, approximately six by two millimeter saline-soaked hemostatic sponge into the exposed cavity above the spinal cord.Then, use 4-O polyglactin 910 sutures to close muscle layers and the skin around the incision site and place the rat in a warm environment under a heat lamp with monitoring until a full recovery. At the appropriate experimental time point for behavioral testing, after the rats have been habituated to the arena, begin the video recording and place a rat in the center of the arena, under dim light conditions to encourage locomotor activity. Allow the rat to explore for at least four minutes, moving the rat back to the center of the arena when it remains stationary for longer than 20 seconds to promote locomotion.Then, score the locomotor performance of the recorded testing session. For articular limb movements, score the hindlimb joint movements during spontaneous locomotion separately for the ankle, knee, and hip, as either normal, slight, or absent. For weight support, evaluate the ability of the hindlimb extensor muscles to contract and support the loaded body weight when the limb is on the ground for when the rat is stationary, as well as during active locomotion.For the digit position, evaluate the position of the hindlimb digits while the rat is stationary and during locomotion. Complete the stepping parameter only if the rat can support its body weight during stepping by rating the orientation of the hindlimb paw placement at the time of the initial contact and at lift off from the ground in addition to the fluidity of the swing phase during stepping. Complete the forelimb/hindlimb coordination parameter only if four consecutive steps occur during testing and if the limbs can actively support body weight.Evaluate the tail position during locomotion as either up or down. Then add the individual scores from each parameter to provide a total for each hindlimb to a maximum of 20 points. To assess and compare lesion sizes between experimental groups, the maximal area of the lesion as a percentage of the total cross-section of the spinal cord can be readily calculated with histological staining of the spinal cord sections.For example, this representative lesion of the left hemicord with an overlay of the proportion of maximal lesion area shared between rats demonstrated the mean lesion size of approximately 47%of the cross-sectional cord area. These representative changes in locomotor performance in the intact state over the first five weeks after left side hemisection demonstrate a significant impairment of locomotion in the left hindlimb of the animal during the first three weeks after the surgery. An improvement to the level of locomotion is observed in the right hindlimb by two weeks post-procedure.The most important things to remember are that an accurate identification of spinal midline for the hemisection and proper post-surgical animal care and monitoring are essential for a successful protocol. This behavioral assessment provides an ideal protocol to screen for upper paretrecorvial indices for supplementing with specialized testing to address specific question of interest on locomotor recovery.

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