This protocol provides a reliable animal model of a spinal cord lateral hemisection that mimics Brown-Sequard syndrome in humans, and a new combined behavior scale for assessing asymmetric behavior deficits. This mouse hemisection model can further our understanding of the neurological impairments that occurs in Brown-Sequard syndrome in humans. Unlike your mentioned assessments, our new combined behavior scale can be applied to any mold that required assessment asymmetrical, locomotive and in-lab deficits.
Using this, consistent, natural hemi-section model can facilitate the comparison of the experimental result between basic and translation research. Using consistent result with this model can be difficult for beginners. The critical step is placing the needle vertically in the midline, as a guidance for inducing the injury.
After watching this video, you should have a good understanding of how to perform both the surgical and the behavioral procedures. On the day of the procedure, clean the surgical table with 70%ethanol, and place a pre-warmed heating pad onto the table. After covering the surgical area with a sterile surgical drape, confirm a lack of response to toe-pinch in an anesthetized rat.
Apply ointment to the animal's eyes to prevent corneal drying, and shave the hair overlying the thoracic vertebrae. Then, clean the exposed skin with three alternating iodine-based and ethanol scrubs. When the animal is ready, locate the thirteenth rib and follow the rib dorsally to identify its connection with the T-13 vertebra.
Count up to identify the T-9 vertebra, and use a number fifteen scalpel blade to make a three to four centimeter midline skin incision over the eight to eleventh vertebral spinous processes. Move the rat under a surgical microscope, and bluntly dissect and separate the paraspinal muscles laterally from the spinous processes towards the facets of the T-9 and T-10 vertebrae on both sides. Disablise the spine with a modified stabilizing holder, make a slit on both sides of the lateral vertebral bone, and slide the stainless steel arms of the holder under the exposed transverse process facets.
Tighten the screws to secure the holder, and use a protractor to expose the T-8 to 11 vertebral laminae and spinous processes. Using a rongeur, snip away the T-9 spinous process, and remove a small portion of the lamina, left to the midline and the entire right portion of the lamina, as laterally as possible. Then insert the ronguer gently under the lamina, and snip one small piece of bone at a time, until the desired region of laminectomy is completed.
Next, identify the dorsal midline of the spinal chord, and insert a 30-gauge needle vertically through the midline, into the spinal chord, all the way to the ventral wall of the vertebral canal, with the bevel facing to the right. Stop any bleeding with a small piece of sterile gel foam, and insert one tip of a pair of iridectomy micro-surgery scissors through the midline of the needle track, and the other tip along the lateral surface of the right hemichord, to make a complete cut on the right hemichord. Use the lateral edge of the same needle as a knife, to cut through the lesion gap to confirm a complete right hemisection.
And visualize the bottom of the vertebral canal, to verify the completeness of the right hemisection. Use cement mixture to build the narrow bridge over the sponge, and the T-8 and T-10 spinous processes. Then, suture the muscle and skin layers separately with 4/0 silk thread, and inject 0.9%sterile saline subcutaneously to maintain hydration.
Pressing the urinary bladder two to three times daily for the first week, and one to two times in the following weeks, until spontaneous bladder voiding returns. At the appropriate experimental time points after the procedure, place the adult rat into an open-field experiment, and examine the animal's locomotion for four minutes. Using the table, assign a value of 1 for yes'and 0 for no'for each behavior category, and sum the total value to give a final unilateral hemisection stepping score of 0-8.
For gate coupling analysis, record the experimental animal walking on a narrow runway device, and assign a score of 0 for no'1 for regular'and/or clumsy'and 2 for normal'for each coupling category. For contact placing assessment, hold the animal in a vertical position, so that both of the hind limbs are available for the placing response, and brush the dorsal surface of one hind limb lightly forward toward the edge of a surface. Observe the foot placement onto the surface, and assign a hind limb contact placing score of 0 for no placement'and 1 for placement'For grid-walking evaluation, place the rat on an elevated thirty six by thirty eight centimeter plastic-coated wire-mesh grid with 3 cm squared openings, and video record the rat walking freely across the platform for 30 steps to allow quantification of the number of foot missteps for each limb to determine the severity of the motor deficit.
Amino fluorescent staining of a cross-section at the injury epicenter shows a complete loss of the right hemichord. And the preservation of the left hemichord contralateral to the injury. Scores of the individual gate assessment measures can be analyzed separately, or they can be combined into a composite combined behavior after hemisection score.
In the first three days post-injury, rats lose the ability to step, with step-like movements beginning to reappear on the ipsilesional side at 7 10 days after injury. And with most steps being dorsal steps. By 28 days after the T-9 hemisection, the rats can take plantar steps with virtually normal coordination.
By comparison, the contralesional hind limb is less interrupted with the unilateral hemisection score dropping within the first five days after the injury, and returning to baseline by day 10, post-injury. For the total gate coupling test, both the stability and adaptability of the coordination after hemisection, are markedly reduced during the first five days after injury. The contact placing and grid-walking of the ipsilateral hind limb are also affected, usually recovering when the animal begins to take plantar steps.
It can be difficult to produce a clean-cut injury. Placing a needle in the middle can be a helpful guideline for insuring a complete separation of the tissue.
