To begin, arrange the sterile surgical equipment on a surgical platform. Approximately 72 hours after the diaphragm muscle electromyographic, or EMG, electrode implantation, weigh the rat. Prior to anesthetizing the rat, place the rat in a Bowman-style cage.
Carefully connect the wires, exiting the rat dorsum to a preamplifier to record bilateral diaphragm muscle EMG. Next, place the anesthetized rat in a prone position. Shave the hair from the neck at about the ear level, and down to the scapulae.
Connect the exposed electrode wires from the rat's back to the amplifier. Disinfect the skin alternating with 4%chlorhexidine gluconate and isopropyl alcohol three times. Cover the rat with sterile surgical covers, except for the upper dorsum.
Using a scalpel, make a four centimeter rostrocaudal incision. Retract the skin and cut the The acromiotrapezius muscle. Then dissect the rhomboid muscle to expose the spinalis muscles.
Under a surgical microscope, remove the spinalis from C1 to C3.Then use rongeur to carefully perform a laminectomy at C2 without damaging major arteries or nerves. Cut and remove the dura mater at C2.While monitoring diaphragm muscle EMG, insert the angle dissecting knife just below the point where the dorsal root enters the spinal cord, and section all the way to the midpoint of the ventral surface. Then record the eupneic ipsilateral diaphragm EMG activity on the anesthetized rat with C2 spinal hemisection performed.
Suture the spinal cord muscles with 3-0 sutures, and then close the skin incision. To maintain hydration, subcutaneously inject one milliliter of saline per 50 grams of animal mass. Place the rat in a clean cage with a heating pad for recovery.
Set the high band-pass filter of the preamplifier to 100 hertz, and the low-pass filter to 1000 hertz. After collecting one to two minutes of left and right hemidiaphragm EMG eupneic recording, save the date in an appropriate format for further analyses. A successful example of C2 spinal hemisection showed the absence of eupneic ipsilateral diaphragm EMG activity under anesthesia on day three post-injury.
Diaphragm muscle EMG activity under awake conditions showed a reduction in eupneic ipsilateral diaphragm compared to the pre-injury baseline. The contralateral diaphragm muscle EMG activity increased in both anesthetized and awake conditions.
