Our research is focused on the plasticity of sympathetic neurons. Some of the questions that we're interested in include how postganglionic sympathetic axons regenerate compared to other neuron types. As well as how sympathetic integration regulates the function of distal tissues that they innervate.
The sympathetic nervous system is well known to be responsible for a wide range of homeostatic mechanisms. More recently, it was discovered that the sympathetic nervous system is also important for maintaining the integrity and function of neuromuscular junctions. This is a fundamental finding that requires a reimagining of the classic nerve-muscle interface.
We've established that postganglionic lumbar sympathetic neurons do not regenerate in response to classic nerve regeneration interventions, such as electrical stimulation and conditioning lesions. There's something innately different about this class of neurons compared to motor and sensory neurons. Using a surgical lumbar sympathectomy allows for anatomically specific ablation of the postganglionic sympathetic system that innervates the lower limbs.
Although chemical sympathectomies are possible, using a surgical method limits off-target effects, as well as the potential for regeneration. Mainstream use of the surgical lumbar sympathectomy has allowed for investigations into the role of sympathetic innervation in exercise-induced metabolic changes in muscle. This protocol can also be adapted to extract these ganglia for neuronal cell culture, as well as for retrograde tracing experiments in postmortem animals.
To begin, position the anesthetized mouse on the surgical table. Cover the mouse with a sterile surgical drape, having a central hole. Using a pair of sharp scissors and fine tipped tweezers, create a midline incision from approximately one millimeter above the level of the pubic synthesis to approximately two millimeters below the ribs.
Identify the midline fascia between the bilateral rectus abdominis muscles. Using the tweezers, lift the abdominal muscles away from the underlying organs and cut along the linear alba to enter the abdominal cavity. Using 5-O sutures, retract the abdominal muscles and skin laterally.
Lay a sterile cotton square saturated in sterile saline on the drape lying superiorly, and to the left of the incision. With two sterile cotton tipped applicators, push the colon and small intestine partially out onto the saline soaked cotton square, ensuring that the cecum and appendix are out of the abdominal cavity. Cover the exposed intestines with another saline saturated cotton square.
Employing closed tweezers, deflect the descending colon to the left to reveal the abdominal aorta and inferior vena cava. Next, with a pair of tweezers, lift the abdominal vessels by surrounding connective tissue. And carefully place a 5-O nylon suture through the connective tissue.
After suture insertion, deflect the abdominal vessels to the left. Using tweezers, bluntly dissect any overlying fascia to visualize a triangle with two sides consisting of the deflected abdominal vessels and the third side defined by the mouse's midline. Next bluntly, dissect any overlying fascia to reveal the ganglia.
Within this triangle, visualize a quadrangle bordered by the left renal artery superiorly, the animal's midline and abdominal vessels laterally. And the left testicular or ovarian artery inferiorly. Carefully blunt dissect with tweezers to enter this quadrangle and identify the bilateral L2 ganglia.
Then using a pair of tweezers, grab the inferior aspect of the visible bilateral ganglia and pull upwards. After achieving the required hemostasis, remove the suture holding the abdominal vessels in place. Using two sterile cotton tipped applicators, carefully replace diverted intestines into the abdominal cavity.
With a 5-O absorbable suture, perform a running stitch to approximate the abdominal muscles. Finally, using 5-O nylon suture, close the skin with simple interrupted stitches. Upon removal of the ganglia, the sweating response due to pilocarpine decreased significantly by postoperative day seven.
Tyrosine hydroxylase staining indicated that surgical lumbar sympathectomy reduced the number of sympathetic axons in the sciatic nerve by postoperative day 21, whereas the neurofilament heavy chain density was largely unaffected.
