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

Summary

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.

Abstract

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.

Introduction

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 primates^3,4. 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 weeks^9,10,11,12. The hemisection model is particularly useful to investigate neural plasticity of damaged and residual tracts and circuits associated with functional recovery^{9,11,12,13,14,15,16,17,18}. 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 rat^2,20. 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 models^10,11,20,22. The assessment score has been used to assess locomotor impairment in the rat following cervical^10,12 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.

Protocol

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 Université de Montréal.

1. Thoracic hemisection surgery

1. 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.
2. Anesthetize the rat under a mixture of isoflurane gas (3% induction, 0.5−3% maintenance) and oxygen (1 L/min). Confirm proper surgical anesthetic depth by verifying the absence of toe pinch 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.
3. 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 °C using a feedback-controlled heating pad monitored by rectal thermometer.
4. Place ophthalmic ointment on the eyes to keep them hydrated and reapply throughout surgery as required.
5. Make a 2.5 cm incision in the skin overlaying the T6−T10 vertebrae with a scalpel. Retract the skin and superficial fat using blunt dissection scissors.
   NOTE: The T6−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 2^nd thoracic vertebra²⁴, or caudally by palpation of the most posterior floating rib which will induce movement in the 13^th thoracic vertebrae.
6. Separate the paravertebral muscles inserting on the dorsal aspect of the T7−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 (~5−15x).
7. 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.
8. 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°), 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.
9. 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−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.
10. 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.
11. 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.
12. 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’s solution (intraperitoneal [i.p.]) immediately after surgery.
13. Remove the rat from anesthesia. Place the rat in a warm environment under a heating pad or lamp (~ 33 °C) until the animal is fully awake.
14. 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.

2. Open-field testing procedure and locomotor performance scoring

1. 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.
2. Place a camera at ground level facing the circular plexiglass open-field arena to record testing sessions for offline analysis (30−60 frames/s minimum).
3. Begin video recording and place the rat in the center of the arena under dim light conditions to encourage locomotor activity.
4. 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.
5. 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).
   1. 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).
   2. 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.
   3. 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.
   4. 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).
      1. 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).
      2. 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.
      3. 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.
   5. 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 (>90% of steps), 2 when frequent (50−90% of steps), 1 when occasional (<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.
   6. 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.
   7. 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 <20 represent increasing amounts of locomotor impairment and a score of 0 indicates limb paralysis.

Results

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...

Discussion

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...

Materials

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