Implantation Surgery for Abdominal Vagus Nerve Stimulation and Recording Studies in Awake Rats

Abdominal VNS has the advantage of not causing cardiac and respiratory side effects associated with cervical VNS. The aim of the study was to investigate the anti-inflammatory effect of abdominal VNS in a rat model of rheumatoid arthritis. Previously, we have shown that abdominal VNS reduces symptoms of a rat model of inflammatory bowel disease.

A first inhuman clinical trial is currently underway to investigate the safety and efficacy of abdominal VNS as a treatment of inflammatory bowel disease. We are expanding our neuromodulation research to investigate whether we can record spontaneous inflammation-induced vagus nerve activity as the next step for developing an adaptive abdominal VNS system for the treatment of inflammatory conditions. We have demonstrated that abdominal VNS causes no cardiac or respiratory off target effects that are associated with cervical VNS.

And it has anti-inflammatory effects in rat models of inflammatory bowel disease and rheumatoid arthritis. The effects of abdominal VNS on the body are not well understood compared to cervical VNS. This protocol may help to establish experimental protocols for testing the effect of abdominal VNS on different physiological parameters in healthy animals, or inflammatory conditions such as systemic lupus and chronic kidney disease.

To begin, place the anesthetized rat on the surgical table. After that, use a 1 milliliter syringe with a 25 gauge needle to administer analgesia subcutaneously. Apply eye gel to the animals to prevent eyes from drying out while under anesthesia.

Then, shave generously around the incision site. Clean the surgical site with Betadine, and place a surgical drape over the animal. Using a 1 milliliter syringe with a 25 gauge needle, administer bupivacaine subcutaneously at the incision site.

Using a scalpel blade, make a 2-centimeter long incision on the back for the percutaneous pedestal. Turn the rat to dorsal recumbency and make a 3-centimeter incision on the skin along the midline just below the xiphoid process. Hold up the skin near the incision site and use dissecting scissors to blunt dissect the skin layer from the muscle layer around the incision.

For subcutaneous tunneling of the electrode array, position the animal on its right side. Insert a hemostat from the ventral incision and blunt dissect towards the dorsal incision site. Next, take a needle cap, cut off its rim, and insert the electrode array for protection during transit.

Tunnel the electrode array under the skin towards the ventral incision with sterile-gloved hands. To assess the esophagus and the vagus nerve, place the animal in dorsal recumbency again. Make a 3-centimeter incision on the muscle layer along the midline below the xiphoid process to expose the liver.

Create a smaller incision on the muscle layer laterally to the main ventral incision. Use the modified needle cap to tunnel the electrode array through this small incision into the abdominal cavity. Then retract the skin and muscle layers to keep the abdominal cavity open.

Using Vannas scissors, cut the surrounding connective tissue and place a small piece of saline-soaked gauze in the abdominal cavity. Position a retractor between the esophagus and the stomach to gently retract the stomach and straighten the esophagus and vagus nerve. Now, expose the ventral surface of the esophagus and identify the abdominal vagus nerve and its subbranches, including the hepatic, celiac, and two gastric branches.

Use fine forceps and Vannas scissors to cut the connective tissue securing the abdominal vagus nerve to the esophagus. Dissect the nerve from just above the hepatic and celiac branches towards the diaphragm. Place an electrode array next to the nerve to ensure that enough length of nerve is separated from the connective tissue to accommodate the array.

Once connective tissue has been cleared, pass silk sutures onto the electrode side of the array cuff under the nerve. Open the array cuff and carefully place the nerve into the array channel. Then, securely tie the sutures around the cuff to prevent the nerve from slipping out and trim the sutures after tying.

Use 7-0 silk suture to attach the tab of the array onto the esophagus, securing the array in place and preventing it from twisting. Reposition the liver correctly. Using a 1-milliliter syringe, administer 1 to 2 milliliters of warm sterile saline in the abdominal cavity.

Close the muscle layer with a 3-0 silk suture using the simple running suture technique. Mix secure square knots with at least three throws at both ends to ensure the sutures hold. Using absorbable suture material, close the skin incision with a buried suturing technique to prevent the animal from removing the suture.

Turn the animal to ventral recumbency. Extend the dorsal incision to 4 to 5 centimeters using scissors, and blunt dissect between the muscle and skin layers so that the connector base of the percutaneous connector can sit flat on the muscle layer. Use silk 3-0 suture to make 6 to 8 simple interrupted sutures around the connector base, securing it to the muscle layer underneath.

Close the skin incision with silk 3-0 suture using the horizontal mattress suture technique. At the completion of the surgery, administer Hartmann's solutions subcutaneously. Begin by inserting an electrode array onto the abdominal vagus nerve in a rat.

To perform electrophysiology testing, connect one end of a cable to the percutaneous connector and the other end to a stimulator. Measure the peak voltage at the end of its first phase of the voltage waveform, and calculate the total impedance using Ohm's law. Then connect a pair of electrodes to the stimulator and another to the recording equipment.

Apply bipolar stimulation to generate ECAPs using the reference electrode of the VNS implant for the differential recording of ECAPs. Make two sets of recordings each averaging 50 repetitions using data acquisition and analysis software. Use the data analysis software to measure the peak-to-peak voltage of the waveforms within the analysis window to analyze the ECAP response.