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 and place on the surgical field.</li>
	<li>Anesthetize the rat under a mixture of isoflurane gas (3% induction, 0.5&#8722;3% maintenance) and oxygen (1 L/min). Confirm proper surgical anesthetic depth by verifying the absence of toe pinch&#160;and corneal reflex responses. Continuously monitor the rat during the entire procedure, and adjust the amount of anesthetic delivery as required to maintain surgical anesthetic depth.</li>
	<li>Shave the dorsal trunk between the hip and the neck, place the rat on the surgical field, disinfect the incision site with alcohol wipes and proviodine solution, and maintain core body temperature at 37 &#176;C using a feedback-controlled heating pad monitored by rectal thermometer.</li>
	<li>Place ophthalmic ointment on the eyes to keep them hydrated and reapply throughout surgery as required.</li>
	<li>Make a 2.5 cm incision in the skin overlaying the T6&#8722;T10 vertebrae with a scalpel. Retract the skin and superficial fat using blunt dissection scissors. 
	NOTE: The T6&#8722;T10 vertebral segments can be identified either rostrally by gentle palpation of the dorsal spinal segments from the base of the skull starting from the noticeable protuberance of the 2nd thoracic vertebra24, or caudally by palpation of the most posterior floating rib which will induce movement in the 13th thoracic vertebrae.</li>
	<li>Separate the paravertebral muscles inserting on the dorsal aspect of the T7&#8722;T9 vertebrae using blunt dissection scissors and a self-retaining retractor. Debride and clear any remaining tissue using fine forceps and cotton tipped applicators to expose the spinous processes and vertebral laminae. 
	NOTE: This, and the following steps are greatly aided by microscopic visualization (~5&#8722;15x).</li>
	<li>Carefully cut the facets (zygapophysial joints) bilaterally on the T7 and T8 vertebrae with delicate bone trimmers. Cut the dorsal connective tissue between the T8 and T9 vertebral laminae superficially with a scalpel (1 mm depth) being careful not to injure the underlaying cord.</li>
	<li>Remove the spinous process of the T8 vertebra with bone trimmers. With curved hemostatic forceps carefully clamped on the T7 spinous process, rotate the caudal end of the T8 laminae slightly rostrally (~20&#176;), insert the bone trimmers under the T8 lamina, and make a midline cut extending along the lamina. Continue 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. 
	NOTE: Be careful to remove all bone fragments created from the laminectomy.</li>
	<li>Drip lidocaine (2%, 0.1 mL) in the exposed spinal canal and remove the dura overlaying the T8 spinal segment using fine forceps and iridectomy scissors. Repeat lidocaine administration to the exposed cord and identify the midline of the cord by visualization of a centre line created between the spinous processes extending between exposed T7&#8722;T9 vertebra. 
	NOTE: Along with the spinous processes on T7 and T9, the exposed dorsal root ganglia on T8 can also be used to aid identification of midline and a 30 G needle can be placed in the midline of the cord to aid with the subsequent hemisection.</li>
	<li>Hemisect the spinal cord from midline towards the one side with a dissecting knife. Be careful not to cut through the anterior spinal artery on the ventral side (do not apply firm pressure to the vertebral body). Using iridectomy scissors, carefully cut through any remaining tissue on the lesioned side of the spinal cord to ensure the ventrolateral quadrant is appropriately transected.</li>
	<li>Place sterile saline-soaked hemostatic sponge (~6 x 2 mm) in the exposed cavity above the spinal cord and suture the muscle layers (4-0 polyglactin 910). Next, suture the skin around the incision site.</li>
	<li>Provide adequate analgesic (buprenorphine 0.05 mg/kg subcutaneous [s.c.]), antibiotic (enrofloxacin, 10 mg/kg s.c.), and replenish lost fluids with 5 cc lactated ringer&#8217;s solution (intraperitoneal [i.p.]) immediately after surgery.</li>
	<li>Remove the rat from anesthesia. Place the rat in a warm environment under a heating pad or lamp (~ 33 &#176;C) until the animal is fully awake.</li>
	<li>Provide supplemental analgesia daily over the first 3 post-surgical days and continually monitor for signs of pain, weight loss, improper micturition, infection, problems with wound healing, or autophagia.</li>
</ol>
2. Open-field testing procedure and locomotor performance scoring
<ol>
	<li>Handle rats daily for 1 week and habituate them to the arena for two 5-min sessions prior to testing to acclimatize to being picked up, gently from the mid-trunk, while in the open-field and to ensure measurement reliability during testing.</li>
	<li>Place a camera at ground level facing the circular plexiglass open-field arena to record testing sessions for offline analysis (30&#8722;60 frames/s minimum).</li>
	<li>Begin video recording and place the rat in the center of the arena under dim light conditions to encourage locomotor activity.</li>
	<li>Continue the testing session for 4 min to ensure an adequate amount of locomotor bouts for analysis. Pick up and replace rats in the center of the arena when they remain stationary for longer than 20 s to promote locomotion.</li>
	<li>Score locomotor performance of the recorded testing session by completing the rubric provided in Table 1 according to the parameters in the following subsections. 
	NOTE: It is helpful to score each parameter separately by repeated viewing of the recorded testing session using software that allows for variable playback speed and frame-by-frame analysis (e.g., VLC media player).
	<ol>
		<li>For articular limb movements, score hindlimb joint movements during spontaneous locomotion separately for the ankle, knee, and hip as either normal (more than half of the range of motion, awarded score = 2), slight (less than half of the range of motion, awarded score = 1), or absent (awarded score = 0).</li>
		<li>For weight support, evaluate the ability of hindlimb extensor muscles to contract and support loaded body weight when the limb is on the ground separately for when the rat is stationary as well as during active locomotion. Award a score of 1 when weight support is present and a score of 0 when weight support is absent. 
		NOTE: Stationary weight support is deemed a perquisite for active weight support.</li>
		<li>For digit position, evaluate the position of the hindlimb digits while the rat is stationary and during locomotion. Award a score of 2 when hindlimb digits are extended, spaced apart from one another, and tonic during locomotion in more than 50% of the testing period (considered normal). Award a score of 1 when digits remain predominantly flexed and a score of 0 when digits remain predominantly atonic.</li>
		<li>For stepping, complete this parameter only if the rat can support its body weight during stepping. Evaluate stepping by rating the orientation of hindlimb paw placement at the time of initial contact and at lift off from the ground in addition to the fluidity of the swing phase during stepping. 
		NOTE: There are 3 scores for this parameter described in the following subsections separately evaluating: 1) the axial orientation of paw placement at limb contact (dorsal/plantar placement), 2) the longitudinal orientation of paw placement at initial contact and during lift (parallel to the body axis or rotated internally/externally), and 3) the quality of limb movement during swing (regular or irregular).
		<ol>
			<li>For the paw placement at limb contact, score the axial orientation of the paw placement at limb contact as 0 when dorsal placements occur in more than 50% of steps. 
			NOTE: Plantar placement is deemed a perquisite for scoring the orientation of paw at contact and lift (step 2.5.4.2), swing movement (step 2.5.4.3) and forelimb-hindlimb coordination (step 2.5.5).</li>
			<li>For the paw orientation at limb contact and lift, award a score of 2 when the longitudinal paw and body axes are parallel and a score of 1 when the limb is rotated externally or internally, separately for both limb contact and lift.</li>
			<li>For the swing movement, award a score of 2 when hindlimb joints move in a harmonious and regular way during swing and a score of 1 when jerky or spasmodic movements of the joints occur during swing.</li>
		</ol>
		</li>
		<li>For forelimb-hindlimb coordination, complete this parameter only if 4 consecutive steps occur during testing and if the limbs can actively support body weight. Award a score of 3 when coordination is consistent (&#62;90% of steps), 2 when frequent (50&#8722;90% of steps), 1 when occasional (&#60;50% of steps), or 0 when absent (0% of steps). 
		NOTE: Forelimb-hindlimb coordination is defined as a regular alternation in stepping between the hindlimb being scored and the forelimb on the same side of the body.</li>
		<li>For tail position, evaluate the tail position during locomotion as either up (off the ground, awarded score = 1) or down (touching the ground, awarded score = 0). 
		NOTE: An elevated tail position during locomotion is an indicator of trunk stability in the rat. After hemisection, the tail is normally held close to or touching the ground as trunk stability is impaired.</li>
		<li>Add the individual scores from each parameter to provide a total for each hindlimb of a maximum of 20 points. 
		NOTE: A score of 20 indicates normal locomotor performance. Scores &#60;20 represent increasing amounts of locomotor impairment and a score of 0 indicates limb paralysis.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

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 beneficial to stabilize the cord delicately with fine forceps placed on either side during the procedure. Placing the rat in a stereotaxic frame with the tail gently taped under light tension can help with stability and proper vertebral alignment during the procedure. A spinal clamp attached to the stereotaxic frame and a spinous process can also be used to enhance stability of the vertebral column, but we find that its presence can restrict access to the cord with surgical tools and requires awkward approach angles during the surgery. It is also essential to remove any bone fragments left in the spinal canal from the laminectomy as they can cause unwanted compression injury to the cord and promote secondary damage.
Rats should be constantly observed during the surgery to monitor necessary vital signs such as core temperature and breathing, as hypothermia is a leading cause of mortality both during anesthesia administration and initially after surgery. Regulation of core body temperature with a rectal probe and feedback-controlled heating pad can greatly avoid temperature complications. A pulse oximeter can also be used to monitor blood oxygenation and heart rate to regulate anesthetic depth. We find that fluid replenishment immediately after surgery with lactate ringer&#8217;s solution warmed to body temperature results in a more rapid recovery time for the rat to awaken after surgery, regain autonomic control of body temperature, and be able to drink and eat.
Post-surgical monitoring of the rat is essential after the hemisection surgery, especially for signs of improper micturition, pain, infection, weight loss, problems with wound healing, or autophagia. Consultation with veterinary staff for evaluation and treatment is crucial in situations of post-surgical complications. In particular, acute spinal shock or unintended bilateral lesions may interfere with micturition that can lead to potentially fatal infections. Carefully monitor the bladder of the rat after surgery and manually void three times per day if full by gentle pressure from the ventral side of the bladder descending caudally. We use female Long-Evans rats as they have a significantly shorter and straighter urethra than males that leads to a more rapid onset of an automatic urinary bladder, easier micturition, and lower rates of urinary tract infections2. Weights should also be monitored and a loss &#62;20% from baseline warrants investigation into food and water intake. The teeth should be checked for malocclusion, the abdomen for ileus, and rats given appropriate supplementary fluids and nutrition such as hydrogel or a liquid diet. A cyst may rarely form under the incision site that can be drained safely with a syringe without complication in consultation with veterinary staff.
The Martinez open-field locomotor assessment procedure provides a simple technique that does not require any specialized equipment, preoperative training, or food deprivation of the animal to perform. The assessment can be performed as early as the animal recovers from anesthesia and can be used to screen animals for appropriate recovery indices (e.g., recovery of body weight support) when more rigorous and specific locomotor testing can be supplemented such as automated gait assessment of overground locomotion26,27,28, kinematic analyses during treadmill locomotion29,30,31,32, grid walking33, and ladder rung walking9,34. Importantly, while the BBB scale has been shown to not be linear with locomotor recovery as scores tend to cluster around certain values19, the Martinez open-field locomotor assessment provides a linear scoring profile during the recovery process10. To ensure reliable behavioral data, it is important to minimize the number of confounders during testing and analysis. To help reduce variability during testing, sessions should occur at the same time of day, in the same room, and by the same experimenter. The open-field assessment can be reliably performed over repeated sessions9,10,11,12,23, but rats may become habituated to the environment over time and reduce their activity during testing resulting in an inadequate amount of locomotor bouts for analysis. To overcome immobility during testing, rats that remain stationary for longer than 20 seconds are picked up and replaced in the center of the arena to promote locomotion. Additionally, including a conspecific in the arena during testing that is marked for identification can help promote locomotor activity in the test rat. To ensure reliability in locomotor scoring two raters, preferably blinded, should conduct the analyses as previously described10.
In conclusion, we describe methods for conducting a thoracic spinal cord hemisection in the rat and assessing spontaneous hindlimb locomotor performance in an open-field arena. Although a procedure for conducting lateral hemisections was described, the technique can be readily adapted to perform either dorsal hemisections35, staggered alternating hemisections36,37, or full transections38 depending on the desired lesion location and amount of spared descending supraspinal innervation. Importantly, the technique can also be used in larger animal models, including cats39,40,41 and non-human primates6,42 with comparable deficits observed between small and large animals, making it useful for investigating both the neurobiological mechanisms of recovery and for preclinical therapeutic testing.

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.

<table><tbody><tr><td>Baytril</td><td>CDMV</td><td>11242</td><td></td></tr><tr><td>Blunt dissection scissors</td><td>World Precision Instruments</td><td>503669</td><td></td></tr><tr><td>Buprenorphine hydrochoride</td><td>CDMV</td><td></td><td></td></tr><tr><td>Camera lens</td><td>Pentax</td><td>C31204TH</td><td>12.5-75mm, f1.8, 2/3&quot; format, C-mount</td></tr><tr><td>CMOS video camera</td><td>Basler</td><td>acA2000-165uc</td><td>2/3&quot; format, 2048 x 1088 pixels, up to 165 fps, C-mount, USB3</td></tr><tr><td>Compressed oxygen gas</td><td>Praxair</td><td></td><td></td></tr><tr><td>Cotton tipped applicators</td><td>CDMV</td><td>108703</td><td></td></tr><tr><td>Delicate bone trimmers</td><td>Fine Science Tools</td><td>16109-14</td><td></td></tr><tr><td>Dissecting knife</td><td>Fine Science Tools</td><td>10055-12</td><td></td></tr><tr><td>Dumont fine forceps (#5)</td><td>Fine Science Tools</td><td>11254-20</td><td></td></tr><tr><td>Ethicon Vicryl 4/0 Violet Braided FS-2&amp;nbsp; suture (J392H)</td><td>CDMV</td><td>111689</td><td></td></tr><tr><td>Feedback-controlled heating pad</td><td>Harvard Apparatus</td><td>55-7020</td><td></td></tr><tr><td>Female Long-Evans rats</td><td>Charles River Laboratories</td><td>Strain code: 006</td><td>225-250g</td></tr><tr><td>Gelfoam</td><td>CDMV</td><td>102348</td><td></td></tr><tr><td>Curved hemostat forceps</td><td>Fine Science Tools</td><td>13003-10</td><td></td></tr><tr><td>Hot bead sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td></td></tr><tr><td>Hydrogel</td><td>70-01-5022</td><td>Clear H20</td><td></td></tr><tr><td>Isofluorane</td><td>CDMV</td><td>118740</td><td></td></tr><tr><td>Lactated Ringer&#039;s solution</td><td>CDMV</td><td>116373</td><td></td></tr><tr><td>Lidocaine (2%)</td><td>CDMV</td><td>123684</td><td></td></tr><tr><td>Needle 30 ga</td><td>CDMV</td><td>4799</td><td></td></tr><tr><td>Open-field area</td><td>Custom</td><td></td><td>Circular Plexiglas arena 96 cm diameter, 40 cm wall height</td></tr><tr><td>Opthalmic ointment</td><td>CDMV</td><td>110704</td><td></td></tr><tr><td>Personal computer&amp;nbsp;</td><td></td><td></td><td>With USB3 connectivity to record video with the listed camera</td></tr><tr><td>Physiological saline</td><td>CDMV</td><td>1399</td><td></td></tr><tr><td>Proviodine</td><td>CDMV</td><td>4568</td><td></td></tr><tr><td>Rodent Liquid Diet</td><td>Bioserv</td><td>F1268</td><td></td></tr><tr><td>Scalpal blade #11</td><td>CDMV</td><td>6671</td><td></td></tr><tr><td>Self-retaining retractor</td><td>World Precision Instruments</td><td>14240</td><td></td></tr><tr><td>Vannas iridectomy spring scissors</td><td>Fine Science Tools</td><td>15002-08</td><td></td></tr><tr><td>Veterinary Anesthesia Machine and isofluarane vaporizer</td><td>Dispomed</td><td>975-0510-000</td><td></td></tr><tr><td>VLC media player</td><td>VideoLAN</td><td></td><td>videolan.org/vlc</td></tr></tbody></table>

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 size of 47.3% &#177; 4.0% of the cross-sectional cord area (n = 6).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59738/59738fig1.jpg" /> 
Figure 1: Representative spinal lesions. (A) Microphotograph of a coronal spinal section at the lesion epicenter from a hemisected rat stained with cresyl violet (cell bodies, purple) and luxol fast blue (myelin, blue) indicating damage to the grey and white matter concentrated in the left hemicord. D, dorsal; V, ventral; L, left; R, right. Scale bar: 1 mm. (B) Schematic overlay of the shared proportion of maximal lesion area in a group of rats (n = 6). The location of the crossed corticospinal tract in the dorsal funiculus on the right side is shaded in black. <a href="https://www.jove.com/files/ftp_upload/59738/59738fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The primary consequence of the hemisection is an initial paralysis of the hindlimb on the side of the lesion during the first two to three postoperative days. Locomotor performance of the more affected hindlimb improves rapidly in the rat after hemisection over the first few weeks after injury. Small deficits in the opposite hindlimb are commonly observed initially after the hemisection that can reflect compensation for the more affected limb, or deficits resulting from a lack of postural stability, weight support, and consistent stepping. A large and persisting deficit in the opposite hindlimb would indicate a bilateral lesion extending into the opposing hemicord.
A sample locomotor performance scoring rubric is provided in Table 1.
Table 1: Sample scoring sheet. Sample locomotor performance scoring rubric. For each parameter, the possible scores are indicated in parentheses. I, internal; E, external; P, parallel; FL-HL, forelimb-hindlimb. <a href="https://www.jove.com/files/ftp_upload/59738/59738_Table1_Revised.xlsx">Please click here to download this file.</a>
The time course of representative changes in locomotor performance in the intact state and over the first five weeks after a left side hemisection in separate groups of rats (n = 6 per group) is depicted in Figure 2.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59738/59738fig2v2.jpg" /> 
Figure 2: Representative time course of changes in hindlimb locomotor performance in the open-field in the intact state and for five weeks after a left side thoracic hemisection. Performance of the left hindlimb (A) is significantly impaired from intact values during the first three weeks after hemisection, and of the right hindlimb (B) during the first week after hemisection. Data are plotted as group mean &#177; standard deviation (SD; n = 6 per group). Statistical analyses were performed with Kruskal-Wallis non-parametric tests supplemented with Dunn&#8217;s multiple comparison tests to assess group differences between time points. *p &#60; 0.05, ***p &#60; 0.001. <a href="https://www.jove.com/files/ftp_upload/59738/59738fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

thoracic-spinal-cord-hemisection-surgery-open-field-locomotor

Universidad Científica del Sur

Research

JoVE Journal

Neuroscience

9.5K Views.  Université de Montréal. 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.

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

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Medicine

Using Laser Doppler Imaging and Monitoring to Analyze Spinal Cord Microcirculation in Rat

Laser Doppler flowmetry (LDF) is a noninvasive method for blood flow (BF) measurement, which makes it preferable for measuring microcirculatory alterations of the spinal cord. In this article, our goal was to use both Laser Doppler imaging and monitoring to analyze the change of BF after spinal cord injury. Both the laser Doppler image scanner and the probe/monitor were being employed to obtain each readout. The data of LDPI provided a local distribution of BF, which gave an overview of perfusion around the injury site and made it accessible for comparative analysis of BF among different locations. By intensely measuring the probing area over a period of time, a combined probe was used to simultaneously measure the BF and oxygen saturation of the spinal cord, showing overall spinal cord perfusion and oxygen supply. LDF itself has a few limitations, such as relative flux, sensitivity to movement, and biological zero signal. However, the technology has been applied in clinical and experimental study due to its simple setup and rapid measurement of BF.

Here we present a combination of laser Doppler perfusion imaging (LDPI) and laser Doppler perfusion monitoring (LDPM) to measure spinal cord local blood flows and oxygen saturation (SO2), as well as a standardized procedure for introducing spinal cord trauma on rat.

Laser Doppler flowmetry (LDF) is a noninvasive method for blood flow (BF) measurement, which makes it preferable for measuring microcirculatory ...

Behavior

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Experimental Strategies to Bridge Large Tissue Gaps in the Injured Spinal Cord after Acute and Chronic Lesion

After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the spinal cord, tumor resections, or tissue defects resulting from traumatic accidents can aid in facilitating general tissue repair as well as regenerative growth of nerve fibers into and beyond the affected area. Two experimental treatment strategies are presented: (1) implantation of a novel microconnector device into an acutely and completely transected thoracic rat spinal cord to readapt severed spinal cord tissue stumps, and (2) polyethylene glycol filling of the SCI site in chronically lesioned rats after scar resection. The chronic spinal cord lesion in this model is a complete spinal cord transection which was inflicted 5 weeks before treatment. Both methods have recently achieved very promising outcomes and promoted axonal regrowth, beneficial cellular invasion and functional improvements in rodent models of spinal cord injury.
The mechanical microconnector system (mMS) is a multi-channel system composed of polymethylmethacrylate (PMMA) with an outlet tubing system to apply negative pressure to the mMS lumen thus pulling the spinal cord stumps into the honeycomb-structured holes. After its implantation into the 1 mm tissue gap the tissue is sucked into the device. Furthermore, the inner walls of the mMS are microstructured for better tissue adhesion.
In the case of the chronic spinal cord injury approach, spinal cord tissue - including the scar-filled lesion area - is resected over an area of 4 mm in length. After the microsurgical scar resection the resulting cavity is filled with polyethylene glycol (PEG 600) which was found to provide an excellent substratum for cellular invasion, revascularization, axonal regeneration and even compact remyelination in vivo.

Severe spinal cord injuries often result in tissue defects. Two possibilities are described to successfully bridge such gaps to promote tissue adaptation, regenerative responses and functional improvement in rats via implantation of a mechanical microconnector system after acute injury and five weeks after complete spinal cord transection.

After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the ...

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human Brown-S&#233;quard syndrome&#160;is a common type of incomplete SCI caused by a lesion to one half of the spinal cord which results in paralysis and loss of&#160;proprioception&#160;on the same (or ipsilesional) side as the injury, and loss of pain and temperature sensation on the opposite (or contralesional) side. Adequate methodologies for producing a spinal cord lateral hemisection (HX) and assessing neurological impairments are essential to establish a reliable animal model of Brown-S&#233;quard syndrome. Although lateral hemisection model plays a pivotal role in basic and translational research, standardized protocols for creating such a hemisection and assessing unilateralized function are lacking. The goal of this study is to describe step-by-step procedures to produce a rat spinal lateral HX at the 9th thoracic (T9) vertebral level. We, then, describe a combined behavior scale for HX (CBS-HX) that provides a simple and sensitive assessment of asymmetric neurological performance for unilateral SCI. The CBS-HX, ranging from 0 to 18, is composed of 4 individual assessments which include unilateral hindlimb stepping (UHS), coupling, contact placing, and grid walking. For CBS-HX, the ipsilateral and contralateral hindlimbs are assessed separately. We found that, after a T9 HX, the ipsilateral hindlimb showed impaired behavior function whereas the contralateral hindlimb showed substantial recovery. The CBS-HX effectively discriminated behavioral functions between ipsilateral and contralateral hindlimbs and detected temporal progression of recovery of the ipsilateral hindlimb. The CBS-HX components can be analyzed separately or in combination with other measures when needed. Although we only provided visual descriptions of the surgical procedures and behavioral assessments of a thoracic HX, the principle may be applied to other incomplete SCIs and at other levels of the injury.

Here we describe surgical procedures to produce a reliable spinal cord lateral hemisection (HX) at the 9th thoracic level in adult rats and neurobehavioral assessments designed for detecting asymmetric deficits after such a unilateral injury.

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human ...

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) ...

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from SCI. Most pre-clinical animal trials are conducted in rodent models of SCI. While rat models of SCI have been widely used, mouse models have received less attention, even though mouse models can have significant advantages over rat models. The small size of mice equates to lower animal maintenance costs than for rats, and the availability of numerous transgenic mouse models is advantageous for many types of studies. Inducing repeatable and precise injury in the animals is the primary challenge for SCI research, which in small rodents requires high-precision surgery. The transection-type injury model has been a commonly used injury model over the last decade for transplantation-based therapeutic research, however a standardized method for inducing a complete transection-type injury in mice does not exist. We have developed a surgical protocol for inducing a complete transection type injury in C57BL/6 mice at thoracic vertebral level 10 (T10). The procedure uses a small tip drill instead of rongeurs to precisely remove the lamina, after which a thin blade with rounded cutting edge is used to induce the spinal cord transection. This method leads to reproducible transection-type injury in small rodents with minimal collateral muscle and bone damage and therefore minimizes confounding factors, specifically where behavioral functional outcomes are analyzed.

This protocol describes how to create a precise laminectomy for induction of stable transection-type spinal cord injury in the mouse model, with minimal collateral damage for spinal cord injury research.

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...

Automated Gait Analysis to Assess Functional Recovery in Rodents with Peripheral Nerve or Spinal Cord Contusion Injury

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and a lot of comparative data has been published in the literature. This includes a multitude of assessment methods to study functional recovery following nerve injury and repair. Besides evaluation of nerve regeneration by means of histology, electrophysiology, and other in vivo and in vitro assessment techniques, functional recovery is the most important criterion to determine the degree of neural regeneration. Automated gait analysis allows recording of a vast quantity of gait-related parameters such as Paw Print Area and Paw Swing Speed as well as measures of inter-limb coordination. Additionally, the method provides digital data of the rats&#8217; paws after neuronal damage and during nerve regeneration, adding to our understanding of how peripheral and central nervous injuries affect their locomotor behavior. Besides the predominantly used sciatic nerve injury model, other models of peripheral nerve injury such as the femoral nerve can be studied by means of this method. In addition to injuries of the peripheral nervous systems, lesions of the central nervous system, e.g., spinal cord contusion can be evaluated. Valid and reproducible data assessment is strongly dependent on meticulous adjustment of the hard- and software settings prior to data acquisition. Additionally, proper training of the experimental animals is of crucial importance. This work aims to illustrate the use of computerized automated gait analysis to assess functional recovery in different animal models of peripheral nerve injury as well as spinal cord contusion injury. It also emphasizes the method&#8217;s limitations, e.g., evaluation of nerve regeneration in rats with sciatic nerve neurotmesis due to limited functional recovery. Therefore, this protocol is thought to help researchers interested in peripheral and central nervous injuries to assess functional recovery in rodent models.

Automated gait analysis is a feasible tool to evaluate functional recovery in rodent models of peripheral nerve injury and spinal cord contusion injury. While it requires only one setup to assess locomotor function in various experimental models, meticulous hard- and soft-ware adjustment and training of the animals is highly important.

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and ...

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.

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 ...

Standardized Evaluation of DIAm EMG Post-C2SH in Rats to Minimize Variability

Assessing Functional Recovery of Eupneic Diaphragm Activity Following Unilateral Cervical Spinal Cord Hemisection in Rats

Following cSCI, activation of the DIAm can be impacted depending on the extent of the injury. The present manuscript describes a unilateral C2 hemisection (C2SH) model of cSCI that disrupts eupneic ipsilateral diaphragm (iDIAm) electromyographic (EMG) activity during breathing in rats. To evaluate recovery of DIAm motor control, the extent of deficit due to C2SH must first be clearly established. By verifying a complete initial loss of iDIAm EMG during breathing, subsequent recovery can be classified as either absent or present, and the extent of recovery can be estimated using the EMG amplitude. Additionally, by measuring the continued absence of iDIAm EMG activity during breathing after the acute spinal shock period following C2SH, the success of the initial C2SH may be validated. Measuring contralateral diaphragm (cDIAm) EMG activity can provide information about the compensatory effects of C2SH, which also reflects neuroplasticity. Moreover, DIAm EMG recordings from awake animals can provide vital physiological information about the motor control of the DIAm after C2SH. This article describes a method for a rigorous, reproducible, and reliable C2SH model of cSCI in rats, which is an excellent platform for studying respiratory neuroplasticity, compensatory cDIAm activity, and therapeutic strategies and pharmaceuticals.

Author Spotlight: Neuromotor Control and Recovery of Diaphragm Function Following Cervical Spinal Hemisection in Rats

Respiratory complications are the leading cause of death in individuals with cervical spinal cord injury (cSCI). Animal models of cSCI are essential for mechanistic evaluations and pre-clinical studies. Here, we introduce a reproducible method to assess functional recovery of diaphragm muscle (DIAm) activity following unilateral C2 spinal hemisection (C2SH) in rats.

Following cSCI, activation of the DIAm can be impacted depending on the extent of the injury. The present manuscript describes a unilateral C2 hemisection ...

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

Summary

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Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Using Laser Doppler Imaging and Monitoring to Analyze Spinal Cord Microcirculation in Rat

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

Experimental Strategies to Bridge Large Tissue Gaps in the Injured Spinal Cord after Acute and Chronic Lesion

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats

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

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Automated Gait Analysis to Assess Functional Recovery in Rodents with Peripheral Nerve or Spinal Cord Contusion Injury

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

Assessing Functional Recovery of Eupneic Diaphragm Activity Following Unilateral Cervical Spinal Cord Hemisection in Rats