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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Thoracic hemisection surgery
- 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.
- 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.
- 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.
- Place ophthalmic ointment on the eyes to keep them hydrated and reapply throughout surgery as required.
- 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 2nd thoracic vertebra or caudally by palpation of the most posterior floating rib, which will induce movement in the 13th thoracic vertebrae.
- 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).
- 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 underlying cord.
- 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.
- 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 center line created between the spinous processes extending between the 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 in the identification of the midline. A 30 G needle can be placed in the midline of the cord to aid with the subsequent hemisection.
- Hemisect the spinal cord from the 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.
- Place a 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.
- 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.
- 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.
- 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.