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.
