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

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.