Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

The overall goal of this procedure is to identify phrenic motor neurons in mice using intrapleural delivery of fluorophore-conjugated cholera toxin subunit beta. Two techniques are shown in order to target the pleural cavity, a transdiaphragmatic and a transthoracic approach. This method can help answer key questions in the neuroscience field such as the distribution of the phrenic motor neurons, their morphology as well as the extension of the neural network.

The main advantage of the procedure is that the visualization of phrenic motor neurons relies on the accumulation of the fluorescent neuroanatomical tracer that has been retrotransported from diaphragm neuromuscular junctions located in the pleural space avoiding invasive procedure in the central nervous system. Visual demonstration of this method is critical as the steps describing palpation and transthoracic injection are difficult to learn because it is difficult to describe with words the act of feeling with the hand and the dynamic of needle insertion through the thoracic wall. Sterility is essential to the surgery.

Use an autoclave or glass bead sterilizer to clean the tools and prepare a clean bench coat. After anesthetizing the animal, check for the absence of reflex and apply ophthalmic ointment. Then shave the ventral skin for the transdiaphragmatic procedure.

Alternatively, shave the right-sided thoracic skin for transthoracic procedure. Shave well and broad enough to prevent hair getting into the surgical field. After removing the hair, ensure aseptic conditions by scrubbing the skin with alternating application of 70%ethanol and Betadine.

Be careful to scrub from the center of the site toward the periphery. Lastly, ensure that the mouse maintains its body temperature by placing it over a homeothermic pad. For each transdiaphragmatic injection, load 7.5 microliters of CTB solution in a sterile 10 microliter microsyringe with a blunt or short bevel 33 gauge needle.

Then with the mouse supine, place a half an inch thick roll of gauze under its neck. Now using a scalpel blade, incise the ventral skin along the midline from the xiphoid process to the umbilical region. To avoid damage to the underlying organs, keep the skin taut and do not apply too much pressure.

Next, using small scissors, carefully detach the skin from the abdominal muscles around the incision. This will make it easier to close the incision. Now perform a laparotomy.

Open the abdominal cavity using small scissors to make a button-hole incision at the level of the umbilicus. Then incise the abdominal muscles along the white line up to the xiphoid process. To view the abdominal surface of the diaphragm, retract the abdominal muscles using commercial or home-made retractors.

Stretch out both sides of the laparotomy and tape down the retractors to the bench coat. Then using an LED lamp, illuminate the abdominal surface of the diaphragm. Next, use soft tweezers to lift up the xiphoid appendix and pull down the lateral lobes of the liver.

With small scissors, carefully cut off the suspensory ligament without damaging the gallbladder or the diaphragm. Now inject the three target sites in the right hemi-diaphragm to respectively cover the sternal, the medial and the crural regions. To avoid puncturing the lungs, only penetrate one to two millimeters beyond the diaphragm sheet.

Deliver 1/3 of the volume to each of the three sites. To close the laparotomy site, suture the abdominal muscles with interrupted resorbable 4-0 sutures placed every three millimeters. Then staple the skin closed with nine millimeter wound clips placed every five millimeters.

Be certain to tighten the staples to prevent the animal from pulling them off, but not so much that they impair healing. For each transthoracic injection, load a sterile 500 microliter insulin syringe with 20 microliters of CTB solution and use a beveled 27 gauge needle. Now position the mouse lateral decubitus and palpate to find the sixth and seventh ribs under the elbow region.

While an assistant extends the right fore and hind limbs, insert the syringe under the sixth or the seventh rib tangentially toward the cranium and with the bevel down. Then penetrate three millimeters. Next, elevate the needle gently and the rib should lift upward if the needle is well-positioned in the thoracic cavity.

Then stabilize the animal's breathing movements using two fingers to apply light pressure on the chest wall. Now deliver the full bolus of CTB solution in a single injection. Make sure to insert the needle tangentially three to four millimeters in the pleural cavity so that when you raise the needle, the ribs also lift.

If you're not sure, remove the needle and do it again. After the injection, immediately position the mouse on the heating pad in a right lateral decubitus orientation. This will help the tracer to spread on the right side of the pleural cavity and it allows for monitoring of the animal's breathing.

Next, administer one milliliter of sterile saline solution subcutaneously and provide followup injections if the animal appears dehydrated or listless. Also, subcutaneously administer Buprenorphine twice a day for the first two post-operative days to minimize any potential pain. Three to four-month-old male C57 black 6J mice underwent the described procedure.

Three days before, half of these mice underwent a unilateral right-sided C4 spinal contusion or a laminectomy without contusion. One week after the CTB injection, whole spinal cords were harvested for histology. Motor neurons in the ventral horn were identified as a linear column of labeled cells from C3 to C5.These cells were located in the ipsilateral gray matter and looked like motor neurons.

In injured animals, a striking loss of labeled neurons was observed at C4 level together with spinal tissue disruption. Next, the CTB-positive phrenic motor neurons were manually counted in every fifth transverse section. The total number of labeled phrenic motor neurons per hemi-cord was slightly lower when using the transdiaphragmatic approach than the transthoracic approach, but this difference was not significant.

However, the number of labeled neurons were halved following the C4 injury and this was significant. While attempting this procedure, it is important to remember to consider the animal welfare and to take measures to minimize the pain and discomfort. Following this procedure, other methods like anterograde neuroanatomical tracing or transsynaptic tracing studies can be performed in order to answer additional questions regarding the connectivity of phrenic motor neurons and afferent projection fibers from the rostral ventral respiratory group.

Though this method can provide insight into morphological, biochemical, developmental, and functional aspects of phrenic motor neurons, it can also be used to study phrenic circuits in mouse models of amyotrophic lateral sclerosis, spinal muscular atrophy, and spinal cord injury.