Assessing Functional Recovery of Eupneic Diaphragm Activity Following Unilateral Cervical Spinal Cord Hemisection in Rats

Our research focuses on neuromotor control of the diaphragm muscle during breathing. Phrenic motor neurons innervating diaphragm muscle fibers receive descending excitatory input from the brainstem, which is primarily ipsilateral, and, therefore, disrupted by upper cervical spinal cord hemisection or C2SH. Following C2SH, there is spontaneous recovery of ipsilateral diaphragm activity, reflecting neuroplasticity.

A major development is demonstrating the role of brain-derived neurotropic factor or BDNF signaling through its high-affinity TrkB receptor in the recovery of diaphragm activity Following C2SH. A second development is the use of machine learning approaches to facilitate high-throughput analyses of diaphragm activity during numerous respiratory cycles. The lack of reliable, unbiased, high-throughput techniques to evaluate basic elements of diaphragm neuromotor control is a challenge in defining recovery of function.

This becomes especially important when you're dealing with recordings from animals that are not anesthetized. Our cervical spinal hemisection intentionally leaves the ipsilateral dorsal funiculus intact, minimizing limb muscle deficits, but still causing loss of diaphragm activity. This protocol emphasizes validation of the loss of diaphragm activity at the time of surgery, thereby establishing a clear starting point for recovery of diaphragm muscle function.

Further exploring the role of BDNF TrkB signaling in neuroplasticity and recovery of diaphragm neuromotor control after cervical spinal cord injury. 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 pre-amplifier 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 acromiotrapezius muscle.

Then dissect the rhomboid muscle to expose the spinalis muscles. Under a surgical microscope, remove the spinalis muscles from C1 to C3.Then use a 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 pre-amplifier to 100 hertz and the low-pass filter to 1, 000 hertz. After collecting one to two minutes of left and right hemidiaphragm EMG eupneic recording, save the date in an appropriate format for further analysis.

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.