ࡱ> OQNq` bjbjqPqP 4F::2222222F....l$F=$#h!222&nnn&22nnn:|,22 Q&=.. <=R"|""2vTnoDd =FFF* FFF* FFF222222 SPINAL CORD SURGERY Vedrana Perkov and Marija Heffer-Lauc School of Medicine, University J. J. Strossmayer, Osijek, Croatia INTRODUCTION Spinal cord surgery is an experimental approach in a study of spinal cord injury. It could be performed on a variety of vertebrate models: fish, frog, lizard, mouse and rat. Each model possesses certain advantages and disadvantages. A fish nervous system continuously produces new neurons, therefore it can regenerate even long pathways like reticulospinal, but their nervous system morphology differs from any other vertebrate. The development of a frog nervous system is well described what is useful for regeneration studies, but tetraploidia of favorite species (Xenopus laevis) makes frogs unfavorable for genetic studies. A lizard (Gallotia galloti) was one of the first models for regeneration studies, but it has unique and species specific mechanism. A rodent model is similar to human in histopathological and physiological responses, but lesions are disabilitating and require daily care. The four most commonly used experimental models of spinal cord injury are: contusion injury, crush injury, partial (dorsal and lateral hemisection) and complete surgical transection. After surgery animals lose bladder control, may suffer a prolonged period of pain, stress and risk of infection and all demand daily monitoring. Depending on the experimental design behavioral studies follow motor and sensory functions from preoperative state up to weeks after surgery. The most usual is BBB (The Basso Beattie Bresnahan) Locomotor Rating Scale and Open Field Testing. Track tracing with Biotin Dextran Tetrarhodamine 10% (BDA 10,000, molecular probe) injected centrally into the motor cortex, the usual method in screening regeneration, gives the best results if it is performed at least 12 days after surgery. Youll be able to see and practice spinal cord surgery and post operative care, but being able to perform uniform lesions on experimental group of animals require skill gained by prolonged period of training. DESCRIPTION OF THE PRACTICAL Estimated length of procedure is 30 minutes. Preparation of surgical space: Clean the surgical space with soap and water, dry and wipe with 70% ethanol. Pre Operative care: Bladder expression; Ophthalmic ointment (physiological solution or Vaseline); Hair removals over surgical area prior to shaving the animal, please make sure the animal is properly anesthetized. You can squeeze the hind paw or the base of the tail looking for a reflex. If reflex is present then wait a few more minutes and check again; Ear or tail marks be sure that animal is properly tagged (e.g. with ear punch). Spinal cord surgery: Sterilize instruments in glass bead sterilizer then 70% EtOH; Anesthetize animal (we use Foran isofloranum in induction chamber); Test hind limb, corneal reflexes. Do not start surgery unless these reflexes are negative; The animals rate and adequacy of respiration, eye color and response to pain are monitored throughout the surgical procedure; Remove hair on dorsal surface of rodent above and around the incision site; Disinfect the skin (we used 70% EtOH); Make a midline incision with scalpel at least 5-6 cm above and below the surgical site by using a number 11 scalpel blade, an eye blade or scouten knife; Lift fat pad and trim back with scissors to reveal vertebral column; Use scissors to cut through muscle on either side of vertebral column. Make note of fat pad vein and do not disturb it. Use gauze to soak up any blood in region. If a great deal of bleeding is evident, do not proceed until animal has stabilized; Establish vertebral level in the area of the last rib is T12; Remove muscle until column is clearly shown; Use micro rongeurs to laminectomize at proper vertebral level; Use micro rongeurs to clear away any bone that obscures the width of the spinal cord. Be careful not to disturb the spinal cord in any way; Use gauze (or cotton ear swab) to soak up any blood in the region; Spinal cord injury: Transection: use microscissors to cut the spinal cord or scalpel number 15. Gently deflect one stump dorsally in order to verify complete transaction; Hemisection: use microscissors to slit dura open along dorsal midline. Insert scissors straight into spinal cord and bring scissors together to complete the cut; After injury, suture muscles that overly the lesion. Use wound clips to seal incision, being careful to align inner edges of skin to allow complete healing of the incision; When anesthetic wears off put animal in cage with soft bedding. Post Operative care: Bladder expression (minimally twice per day); Antibiotics and analgetics (3-4 days after surgery, once per day); Water bottles should be easily accessed (bottles with long-sipper tubes, water shouldnt leak). Mice typically resume activity (eating, drinking, grooming and mobility within cage) the day following surgery. The lack of hind limbs movements is sign of well performed surgery. Animals which fail to resume normal activities or express some other sign of distress (like wounds to digit due to autophagie caused by lack of sensation, instability, skin lesions) should be euthanized. Bladder expression is required until animal regains bladder control or the animal is euthanized. SUGGESTED LITERATURE: Basso DM, Beattie MS, Bresnahan JC (1996) Graded Histological and Locomotor Outcomes after Spinal Cord Contusion Using the NYU Weight-Drop Device versus Transection. Exp Neurol 139:244-256. Basso DM, Beattie MS, Bresnahan JC, Anderson DK, Faden AI, Gruner JA, Holford TR, Hsu CY, Noble LJ, Nockels R, Perot PL, Salzman SK, Young W (1996) MASCIS Evaluation of Open Field Locomotor Scores: Effects of Experience and Teamwork on Reliability. J Neurotrauma 13(7):343-359. Ferguson AR, Hook MA, Garcia G, Bresnahan J, Beattie MS, Grau JW (2004) A Simple Post Hoc Transformation that Improves the Metric Properties of the BBB Scale for Rats with Moderate to Severe Spinal Cord Injury. J Neurotrauma 21(11):1601-1613. Marques SA, Garcez VF, Del Bel EA, Martinez AMB (2009) A Simple, Inexpensive and Easily Reproducible Model of Spinal Cord Injury in Mice: Morphological and Functional Assessment. J Neurosci Methods 177: 183-193. 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