The overall goal of the experiment is to generate a reproducible laceration lesion of the mouse spinal cord. This is achieved by exposing the cervical lamina posteriorly when the animal is positioned in the Louisville injury system apparatus. As a second step, a specially designed stabilizing device is used for the fixation of the target vertebra of the mouse spine.
Next, the target spinal cord between the laminar arches is identified after removing the ligamentum flavum at the end. The cervical dorsal hemi section at a depth of 0.75 millimeters is precisely created with the use of an oscillating blade and a micro driver. The major advantage of the technique that we've developed in the laboratory is the creation of a very accurate method of producing a laceration spinal cord injury.
This is important because the techniques that have been used thus far that is the scissors or the scalpel, have created problems that are incomplete and inaccurate methods of spinal cord injury. We now have methods to avoid the contusion of the spinal cord, the displacement of the spinal cord away from the lacerating device, and the excessive hemorrhage that occurs in the spinal cord injury. By using this technique, we are able to create a very precise method that avoids the method of visual estimation of the depth of the spinal cord injury.
By using this technique, we feel that we can revolutionize the methods of experimental spinal cord injury used in the laceration model, which may be inherently important in axon regeneration, as well as in the creation of finding a cure for spinal cord injury. Demonstrating this procedure is eing Jang, a neurosurgeon who is a neurosurgical research scientist in our laboratory, Begin this procedure by sterilizing the following surgical instruments, two to three pairs of forceps, three pairs of micro scissors, 30 gauge needle, micro URS suture, and a needle holder skin clips, and a clip applicator. Then disinfect the spine stabilizer after that, anesthetize the mouse with a cocktail of ketamine, xylazine, or tri ethanol intraperitoneal.
Next, shave the hair from the mouse's neck. Clean the skin with a povidone iodine solution and 70%alcohol. Then transfer the mouse onto the operating table with a prewarm heating pad.
Apply ophthalmic ointment to the eyes to prevent corneal drying. After anesthetic induction, make a posterior cervical midline skin incision from the oput to the subcutaneous fat pet of the lower cervical spine. Under the microscope, make a midline skin incision between the trapezius muscles at C two and retract the semifinalist capita muscles laterally.
Next, extend the midline muscle dissection coddly to the T two spinous process that serves as a reliable landmark. Separate the muscles from the T two spinous process. Then remove the cartilaginous portion of the T two spinous process.
Afterward, dissect the paraspinal muscles off the laminar arches bilaterally from C four through T two using a pair of micro scissors. It begins adjacent to the spinous processes and extends over both laminar arches to the facet joints. After the lateral facets are exposed, place the mouse on the U-shaped channel of the mouse spine stabilizer.
Then place the stainless steel arms of the mouse stabilizer beneath the exposed facets bilaterally. Once the arms are in place, tighten the thumb screws of the steel arms to immobilize the spine under the microscope. Remove the ligamentum flam between the C five and C six laminar arches to expose the underlying dura between the interlaminar space.
Use a 30 gauge needle to create a small dur otomy and micro scissors to expand the dur otomy. The spinal cord is now ready to undergo the controlled laceration lesion. In this procedure place the spine stabilizer and the mouse on the Louisville injury system apparatus stage in which a blade is attached with its position controlled by micro drivers.
Capable of three ranges of motion to perform a dorsal HEMI section lesion between the C five to six lamina with a 2.3 millimeter flat blade attached to the oscillator. Set the amplitude of the oscillating blade to 0.5 millimeters as lower amplitudes may diminish the ease of complete cord laceration. Then turn on the blade vibrator switch under the microscope, place the mouse's spinal cord directly beneath the vibrating blade.
Next, elevate the stage towards the oscillating blade with the micro driver control record. The zero position when the blade barely touches the dorsal vein of the spinal cord, the depth of the spinal cord laceration is measured relative to the zero position. Then elevate the stage position with the micro driver control.
A 360 degree turn of the micro driver knob elevates the stage by 0.25 millimeters. Thus, a 0.75 millimeter dorsal hemi section lesion is created by turning the micro driver knob three times as the blade begins to lacerate the spinal cord lubricate the surgical field with saline irrigation. Once the predetermined depth has been reached, turn off the vibrator switch, lower the stage supporting the mouse and the clamp from the cutting blade.
Remove the blood by irrigating with saline over the surgical site. Then dry the area with cotton Q-tips as hemostasis usually occurs within one minute. At the end, remove the mouse from the spine stabilizer and close the skin with stainless steel.
Michelle clips. The cervical spine is fixated by placing the arms under the lateral facets and then locking the thumb screws as shown in this figure, the dura is exposed between the laminate of C five to six C, six to seven, and C seven to T one without any removal of bone. Here shows the sagittal views of four dorsal spinal cord lacerations at the depths of 0.5, 0.8, 1.1, and 1.4 millimeters.
They indicate the high degree of precision using this technique. One of the problems that has been inherent in the creation of spinal cord injuries has been the lack of stabilization of the cervical spine. Using the spine stabilizer that has been developed by our group, we are now able to create an absolute immobile cervical spine.
This will allow enhancement of the spinal cord injury using the spinal cord contusion, spinal cord injections, as well as cell transplantation into the spinal cord for all methods of spinal cord research.
