An Implantable System For Chronic In Vivo Electromyography

This study has been approved by the institutional animal care and use committee (IACUC) of Vanderbilt University and was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, Maryland). This system includes five implantable components and one external cable.

1. Two Bipolar RLN Stimulus Electrode Cuffs, Each with Pair of Coiled Lead Wires and Terminal Pins

1. Use Teflon-coated, multi-filament, type 316 stainless steel wire (with insulated diameter of 0.0078” or 0.198 mm) for each cuff lead wire. Cut a 70 cm length of wire and coil it into a 12 cm long spring using a coiling device or procure prefabricated coiled leads. If necessary, stretch the spring to increase its length for each implant site. Leave the ends of the coiled leads straight at 3 mm and 10 mm lengths and deinsulate them.
2. Solder a gold-plated copper female pin onto the 3 mm end of the coiled lead.
3. To prepare the nerve cuff, cut a 5 mm segment of silicone tube (OD = 0.156”, ID = 0.094”; or OD = 3.96 mm, ID = 2.39 mm) from a roll of the tubing.
4. To insert a lead into the tube, use a 25 G hypodermic needle to pierce through the tubing wall 1.5 mm from the end and off-center close to the inner wall. Backfill the 10 mm end of the lead into the tip of the needle. Withdraw the needle to deposit the deinsulated portion into the tube. Bend back the bare wire end outside the tube and twist onto the lead at its point of entry into the tube.
   NOTE: Use an operating microscope to perform these steps. A probe can be placed into the tube to curve the wire against the inner wall. The goal is to position the bare portion of the wire so that stimuli can be delivered to the nerve without risking mechanical damage to the nerve.
5. Insert the second lead 1.5 mm from the opposite end of the tube using the same procedure. Align the point of entry to that of the first lead. Pierce the wall with the needle so that the bare portion of the wire is deposited near the inner wall opposite to the first lead.
   NOTE: Looking down the tube, the two stimulus electrodes should form a 45° “V” shape, which will straddle the nerve once in place and assure current delivery through the nerve from anode to cathode.
6. Make an S-shaped slit in the tube wall opposite the electrode points of entry using a pair of curved scissors.
   NOTE: The spiral lips of the cuff can then be opened to situate the nerve inside between the electrodes during surgery.
7. Insert a length of 6-0 monofilament, nonabsorbable suture into the cuff wall at each end using a curved microsurgical needle for eventual securement of the cuff around the nerve.
8. Apply medical grade type-A silicone gel to reinsulate all exposed bare wire outside the cuff.

2. Two bipolar SLN Stimulus Electrode Cuffs, Each with Pair of Coiled Lead Wires and Terminal Pins

1. Assemble the SLN stimulus electrode cuff in the same way as the RLN stimulus electrode cuff. However, use the smaller diameter (OD = 0.125”, ID = 0.062”; or OD = 3.18 mm, ID = 1.57 mm) tube, because the nerve is smaller in diameter.

3. Two PCA muscle EMG recording electrodes, each with Coiled Lead Wire and Terminal Pin

1. Assemble a coiled lead for the PCA muscle electrode as done in step 1.1.
2. Solder a female pin onto the lead as done in step 1.2.
3. Insert the 10 mm end of the PCA muscle lead into the tip of a deep brain stimulation (DBS) electrode using the same strategy for needle-lead insertion into a cuff (step 1.4). Bend the end of the lead to form a hook and clip it to provide a total of 5 mm recording length.
   NOTE: In this application, the PCA muscle and its reinnervating nerve terminals are exposed to electrical conditioning. Stimuli are generated by an implantable pulse generator (IPG) and delivered to the laryngeal muscle through a DBS electrode (Figure 1, inset). This system is adapted from therapeutic brain stimulation (e.g., Parkinson’s disease). The DBS electrode will be inserted into a submuscular pocket and anchored in place. If technology for electrical conditioning of the muscle is not required, the PCA EMG electrode can be directly inserted into the muscle and anchored by its hook.

4. Two TA-LCA Muscle Complex EMG Recording Electrodes, Each with Coiled Lead Wire and Terminal Pin

1. Assemble a coiled lead for the TA-LCA muscle electrode as done in step 1.1.
2. Solder a female pin onto the lead as done in step 1.2.
3. Excise a 5 mm x 10 mm rectangular piece of knitted polyester graft. Make a hole in the center of the mesh with a 20 G hypodermic needle. Introduce the 10 mm end of the lead into the hole with an additional 3 mm of coil protruding beyond the hole. Affix the lead to the mesh using 6-0 monofilament, nonabsorbable suture.
   NOTE: This piece of mesh will be used to anchor the electrode lead to the thyroid cartilage overlying the muscle complex.
4. Bend the end of the lead to form a hook and clip it to provide a total of 5 mm recording length.

5. Skin Receptacle for Interfacing Connections Between Electrodes and External Equipment

1. Utilize a single row female pin stripe connector to make the receptacle. Cut two pieces (each 17.5 mm in length) from the strip, each containing eight pin holes. First, roughen the external surfaces of each piece with sandpaper, then glue them together with phenol in a fume hood to make a double-row connector. Place the connector in 60–80 °C water in a fume hood for 30 min to allow for glue hardening.
   NOTE: This double-row assembly format will provide convenience in the assignment of pinholes for left- vs. right-side electrodes.
2. Cut a 25.6 mm length piece from the strip to make the connector’s faceplate (the portion that will protrude outside the implant site for skin anchoring). Cut a 5.4 mm x 17.4 mm rectangular hole in the middle of the faceplate with a scalpel.
3. Place the double-row connector inside the rectangular hole of the faceplate until it is flushed with the faceplate surface without protrusion. If the connector does not fit into the rectangular hole of the faceplate, the hole can be slightly enlarged with a file. Since the connector holes are not symmetrical, insert the connector edge with the larger diameter holes into the faceplate.
   NOTE: As a result, a female pin inserted into the opposite edge of the connector with the smaller diameter hole will snap and lock into place.
4. Use phenol to glue the connector and the faceplate together. Place the assembly in 60–80 °C water in a fume hood for 30 min to allow for glue hardening.
5. Drill a 1.3 mm hole at each corner of the faceplate and on each side of the faceplate halfway from the ends for a total of six holes.
   NOTE: These holes will be used to suture the final skin receptacle at the implant site.
6. Cut a 15 mm length tube of knitted polyester graft to surround the assembly below the faceplate, making the assembly biocompatible. To fix the tube to the assembly, use a hypodermic needle to thread stainless steel wires through the wall at three equally spaced positions (each 3.8 mm apart) along its length.
7. Place equally spaced notches in each corner of the connector to anchor the wires against the assembly surface. Twist the ends of each wire with a pair of pliers to cinch the tube to the assembly to form a skirt.
8. Make a permanent mark on the polyester patch at one end of the receptacle.
   NOTE: Use this mark for orientation to identify the rostral end of the receptacle during implant surgery. In the rostral to caudal direction, the following pin electrode assignment for each of the two rows (left side and right side) should be as follows: PCA EMG, TA-LCA EMG, empty hole, empty hole, RLN anode, RLN cathode, SLN anode, and SLN cathode.

6. External Connection Cable to Recording Pre-amplifier and Stimulator

NOTE: A cable is used for making connections between the implanted skin receptacle and external equipment during nerve stimulation-EMG recording sessions (sections 8 and 10). It is composed of 12 insulated wires terminating with male pins to insert into female pins in the skin receptacle. This cable consists of two parts: an EMG recording plug and nerve stimulation wires. A recording plug is necessary to isolate low voltage EMG signals from higher voltage stimulus artifacts radiating from stimulus pins. For the same reason, two holes in each row of the skin receptacle are left unoccupied to separate recording pins from stimulation pins.

1. To make the EMG recording plug, use a male strip connector (same length and width, but one-half the height of a female connector). Cut it into two pieces, each containing only two holes. Affix the two pieces using phenol adhesive using the same approach to make the double-row connector in the skin receptacle (step 5.1). Take the four EMG recording wires in the cable and insert their terminal male pins into each of the four holes until they lock in place with the tips protruding beyond the strip edge.
2. Use bone cement to seal the top of the plug to insulate wire-pin junctions.
3. Use the remaining eight wires in the cable terminating in male pins to make individual connections to the nerve stimulation cuffs via their female pins.

7. First Implant Surgery

1. Obtain a 1–2 year-old, 20–25 kg canine of either sex from a licensed farm. Acclimate the animal before aseptic implant surgery. Autoclave all equipment before surgery. Withhold food for 10–12 h before the surgery.
2. Prepare the animal for surgery.
   1. Shave the animal’s head and neck and clean the skin with alcohol and betadine scrub solution. Anesthetize the animal by intravenous injection of 2–4 mg/kg tiletamine and zolazepam combination, followed by 3% isoflurane in oxygen through intubation.
   2. Place the animal on an operating table with a heating pad in supine position and surgically drape the animal. Monitor animal’s heart rate, respiratory rate, body temperature, and oxygen saturation at least every 15 min throughout the surgery to ensure physiological stability at a moderate plane of anesthesia.
3. Make a midline neck incision from the thyroid notch to manubrium. Dissect the trachea free from the esophagus and expose the inferior border of the cricoid cartilage.
4. Position the stimulus cuff onto each of the bilateral SLNs and RLNs. Close the lips of each cuff using the enclosed sutures.
5. Make a cartilage window with a biopsy punch (4 mm in diameter) at the anterior surface of the thyroid cartilage on each side. Expose the lateral aspects of both TA-LCA muscle complexes. Insert the EMG recording electrodes into the TA-LCA muscle complexes using a 23 G needle by inserting the barb into the tip of the needle. Suture the electrode polyester patch onto cartilage.
6. Place the DBS electrode along with its companion hook-wire EMG recording electrode underneath the PCA muscle on each side. Use an endoscope to confirm that stimulation produces vocal fold abduction for each channel. Anchor the DBS electrodes to the cricoid cartilage by 4-0 nonabsorbable sutures.
7. Insert all the wire leads of the nerve stimulation-EMG recording electrodes into the receptacle via their female pins. Press the pins into holes with an insertion tool fashioned from a hemostat. Seal the inferior surface of the receptacle to insulate lead-pin junctions using bone cement.
   1. After the cement hardens, place the receptacle at the rostral end of the midline incision through the skin and suture it to subcutaneous tissues via its polyester skirt. Attach the skin edge to the receptacle by sutures passing through the holes in the faceplate.
      NOTE: One jaw of the hemostat has an end slit leading to a counter-sink hole. The lead wire can be positioned through the slit into the hole and the countersink placed against the head of the pin. The second jaw is placed on the opposite side of the receptacle. Squeezing the hemostat presses the pin into its respective receptacle hole.
8. Make an incision on the left neck to expose the trapezius muscle. Perform dissection to make a submuscular pocket for placement of the implantable pulse generator. Tunnel each DBS lead subcutaneously to the neck incision for insertion into the IPG.
9. Close all surgical wounds with sutures. Monitor the animal closely until full recovery from the surgery.
10. Provide postoperative analgesics (e.g., buprenorphine: 0.01–0.02 mg/kg) routinely for up to 48 h. Administer antibiotics (e.g., cefpodoxime: 10 mg/kg) orally to the animal for at least 3 days. House the animal singly thereafter for throughout the study, and restrict exercise for a period of 10 days to allow normal wound healing and stabilization of the implanted device.
    NOTE: The skin receptacle should be cleaned daily with tissue-compatible antiseptic solution. In addition, dummy male pins should be inserted into the female pins of the skin receptacle routinely except during the EMG recording sessions. This maneuver will avoid the accumulation of debris in the receptacle, allow effective connections to be made with the external cable, and prevent infection.

8. Nerve Stimulation-EMG Recording Sessions at Baseline

NOTE: Perform these sessions 2x–3x after implant surgery (section 7) and before nerve transection surgery (section 9) to obtain baseline EMG signals when the bilateral RLNs are intact. Apply the following protocol during a standard nerve stimulation-EMG recording session (sections 8 and 10).

1. Withhold food before the procedure for 10–12 h. Anesthetize the animal with tiletamine and zolazepam combination (initial loading dose 2–4 mg/kg by intravenous injection, then maintain with 0.4 mg/kg per hour via an i.v. line). Place the animal on a heating pad in supine position and maintain the animal in a moderate plane of anesthesia. Monitor the animal’s vitals during the procedure as described in step 7.2.
2. Insert a zero-degree rigid endoscope with an attached CCD video camera through a laryngoscope to visualize vocal fold motion at the level of the glottis.
3. Interface the external cable that connects to the lab stimulator and EMG preamplifiers to the skin receptacle via its plug and pins. Connect the outputs from the preamplifiers to a data acquisition device and/or an oscilloscope to display, record, and measure EMG signals.
4. Deliver stimuli (single square-wave pulses, 0.1–0.5 ms duration, 0.5–2.0 mA amplitude) to the left and right RLNs, respectively, to record evoked EMG responses from bilateral TA-LCA complexes and PCA muscles under each condition.
5. Deliver stimuli (single square-wave pulses, 0.1–0.5 ms duration, 0.5–2.0 mA amplitude) to the left and right SLNs, respectively, to record evoked EMG responses from bilateral TA-LCA complexes and PCA muscles under each condition.
6. Deliver CO₂ mixed with room air through the mouth of the animal to induce hypercapnia and increase the animal’s respiratory effort. Limit exposure to 1 min, during which the maximum inspiratory motor unit recruitment will occur. Record spontaneous EMG activities of TA-LCA complexes and PCA muscles under this hypercapnic condition.
7. Monitor the animal until full recovery from anesthesia and return the animal to the facility.

9. Second Surgery for Nerve Transection and Anastomosis

1. Perform the second surgery 10–14 days after the first surgery. Withhold food for 10–12 h before surgery.
2. Anesthetize the animal, drape and monitor vitals intraoperatively using the technique described in step 7.2.
3. Remove the sutures and reopen the midline incision by blunt dissection whenever possible. Avoid damage to the previous implantation during the dissection. Expose the bilateral RLNs through dissection. Isolate, transect and anastomose each nerve with 7-0 monofilament, nonabsorbable sutures to induce bilateral laryngeal paralysis.
4. Irrigate the neck incision with sterile saline and gentamycin antibiotic. Close the muscular and subcutaneous tissues using 3-0 absorbable sutures. Close skin with 3-0 nonabsorbable monofilament sutures.
5. Closely monitor the animal until full recovery from surgery.
6. Provide analgesics (e.g., buprenorphine: 0.01–0.02 mg/kg) routinely for up to 48 h postoperatively. Give antibiotics (e.g., cefpodoxime: 10 mg/kg) orally to the animal for at least 3 days. Restrict the animal from exercise for a period of 10 days to allow normal wound healing.

10. Nerve Stimulation-EMG Recording Sessions Following Bilateral RLN Injuries

1. Perform these sessions 1x per week during the first 3 months, then biweekly thereafter. Follow the protocol described in section 8 for these sessions.