TITLE: Carbon nanotube (CNT) yarn fabrication for chronic interfacing with the autonomic nervous system
AUTHORS: Grant Mccallum, Xiaohong Sui, Chen Qiu, Joseph Marmerstein, Yang Zheng, Thomas Eggers, Chuangang Hu, Liming Dai, Dominique Durand

[DESCRIPTION]
The ability to reliably and safely communicate chronically with small diameter (100–300 μm) autonomic nerves could have a significant impact in fundamental biomedical research and clinical applications. However, this ability has remained elusive with existing neural interface technologies. Here we show a new chronic nerve interface using highly flexible materials with axon-like dimensions. The interface was implemented with carbon nanotube (CNT) yarn electrodes to chronically record neural activity from two separate autonomic nerves: the glossopharyngeal and vagus nerves. The recorded neural signals maintain a high signal-to-noise ratio (>10 dB) in chronic implant models. We further demonstrate the ability to process the neural activity to detect hypoxic and gastric extension events from the glossopharyngeal and vagus nerves, respectively. These results establish a novel, chronic platform neural interfacing technique with the autonomic nervous system and demonstrate the possibility of regulating internal organ function, leading to new bioelectronic therapies and patient health monitoring.

[STEPS]
SECTION: Carbon nanotube (CNT) yarn fabrication
1. VA-MWCNT arrays (with MWCNTs of ca.250 μm in length andca.30 nm in diameter) (Fig. 1a), were first synthesized on Fe/Al-coated Si/SiO2 wafers using a low pressure chemical vapor deposition (CVD) method according to our previously reported procedure52.
SECTION: Carbon nanotube (CNT) yarn fabrication
2. A 10-nm thick Al layer was coated on the silicon wafers before the deposition of 3-nm thick Fe film.
SECTION: Chronic In vivo implantation procedure
17. All surgical and experimental procedures were done with the approval and oversight of the Case Western Reserve University, Institutional Animal Care and Use Committee to ensure compliance with all federal, state and local animal welfare laws and regulations.
SECTION: Chronic autonomic nerve recordings
35. The recording system was designed to accommodate two, differential recording channels. Each recording channel has eight amplifier hardware averaging for increased signal-to-noise recording results53. This was achieved by creating, two custom, printed circuit boards (PCB) (Supplementary Fig. 4a and b).
SECTION: Carbon nanotube (CNT) yarn fabrication
3. The catalyst coated substrate was then inserted into a quartz tube furnace for the VA-MWCNT array growth using high-purity (99.99%) acetylene as the carbon source and Ar/H2(2.5:1 v/v) as a carrier gas under 0.2 atm pressure at 760 °C52.
SECTION: Carbon nanotube (CNT) yarn fabrication
4. For spinning VA-MWCNT arrays into the CNT yarn (Fig. 1a), one piece of the VA-MWCNT array of approximately 3-mm width on Si/SiO2 wafer was initially peeled off from the edge of the sidewall, drawn away from the CNT array, and attached onto a tip of the rod, which was connected to a motor27.
SECTION: Carbon nanotube (CNT) yarn fabrication
5. A CNT yarn was spun by the rotating rod that was moved away from the array, and at the same time the bundle was rotated anti-clock-wise to form the CNT yarn (Supplementary Fig. 1b). The rotating speed was in the range of 2000–20000 rpm and the diameter of the as-prepared yarn was in the range of 10–20 μm, which was controlled by the number of CNTs initially pulled out from the array. The yarn diameter was determined by SEM images.
SECTION: CNT yarn electrode assembly process
6. The implant electrode assembly is comprised of two different wire types: CNT yarn for implantation in the nerve and a more robust lead wire used to route the electrode under the skin to a percutaneous electrical connector.
SECTION: CNT yarn electrode assembly process
6.1. The electrode lead wire is a 25 cm long (1 × 7 × 25.4 µm: 127 µm diameter) metal-to-metal composite wire with a silver core (28%) and a stainless steel outer tubing coated with PFA insulation.
SECTION: CNT yarn electrode assembly process
6.2. The DFT® wire insulation was stripped by approximately 3 mm at one end and joined to the CNT yarn by conductive epoxy resin.
SECTION: CNT yarn electrode assembly process
7. These wire assemblies were then placed and fixed on a custom-made acrylic rack (Supplementary Fig. 2a) for insulating the CNT yarn wire and junction.
SECTION: CNT yarn electrode assembly process
8. Parylene-C was chosen to be the insulation material for the CNT yarn. The Parylene-C thickness was 3.5 µm and was deposited using chemical vapor deposition (CVD).
SECTION: CNT yarn electrode assembly process
8.1. During the CVD coating process, a 500 µm portion of the CNT yarn end was left uninsulated by using tape to mask the desired area.
SECTION: CNT yarn electrode assembly process
8.2. After the Parylene-C insulation was deposited, the individual electrode wire assemblies were removed from the rack.
SECTION: CNT yarn electrode assembly process
9. The junction between the CNT yarn and the DFT® lead wire was placed within a 6 mm long (0.51 mm ID, 0.94 mm OD) silicone tube.
SECTION: CNT yarn electrode assembly process
10. Any empty space inside the silicone tube was filled with silicone elastomer to further insulate the wire-to-wire junction.
SECTION: CNT yarn electrode assembly process
11. The uninsulated CNT yarn end and a short portion of the insulated yarn was wound around the tip of a Tungsten microneedle
SECTION: CNT yarn electrode assembly process
12. The silicone tube between the CNT yarn and DFT® wire was fixed to the microneedle using a slip knot made of thread (Supplementary Fig. 2b).
SECTION: CNT yarn electrode assembly process
13. After winding, 3 mm of DFT® wire insulation at the free end was stripped to be eventually soldered to a percutaneous electrical connector.
SECTION: CNT yarn electrode assembly process
14. A ground electrode (Supplementary Fig. 2c) was made with a 3 × 4 cm, 25 µm diameter Platinum-Iridium (Pt-Ir) wire and DFT® wire.
SECTION: CNT yarn electrode assembly process
15. The Pt-Ir wire and DFT® wire were joined together by conductive epoxy.
SECTION: CNT yarn electrode assembly process
16. This ground electrode was placed chronically under the dorsal skin of the animal.
SECTION: Chronic In vivo implantation procedure
18. Adult male Sprague Dawley rats (350–450 grams, Charles River Laboratories) were used for all peripheral nerve implants discussed.
SECTION: Chronic In vivo implantation procedure
19. Prior to surgery, all surgical instruments and implant assemblies were autoclave sterilized at 250 °C for 1 hour.
SECTION: Chronic In vivo implantation procedure
20. During surgery, the animals were gas anesthetized under 2% isoflurane in 1 L/min oxygen.
SECTION: Chronic In vivo implantation procedure
21. For each of the nerves, a section approximately 3–4 mm was completely separated from and devoid of all connective tissue.
SECTION: Chronic In vivo implantation procedure
22. A custom made hook (Fig. 1c, Supplementary Fig. 3a) was attached to a three-axis micromanipulator and positioned to provide a small amount of tension and suspend the nerve in the air.
SECTION: Chronic In vivo implantation procedure
23. The slip knot was then removed, thereby releasing the silicone tube junction from the microneedle.
SECTION: Chronic In vivo implantation procedure
24. The CNT yarn electrode assembly was then advanced into the nerve until all of the uninsulated CNT yarn segment was within the perineurium.
SECTION: Chronic In vivo implantation procedure
25. Micro tweezers were then used to apply a small amount of pressure at the implant site while the Tungsten microneedle was carefully removed leaving the CNT yarn inside the nerve (Supplementary Fig. 3b).
SECTION: Chronic In vivo implantation procedure
26. The hook was then advanced along the nerve to the next implant site, approximately 2 mm away, and the implant process was repeated.
SECTION: Chronic In vivo implantation procedure
27. The CNT yarns and the silicone tube junctions were then carefully positioned within the incision site and completely encapsulated with 1 mL of fibrin glue (Supplementary Fig. 3c) to secure the implant in place while the normal physiological process of collagen encapsulation took place while the fibrin glue was absorbed by the body.
SECTION: Chronic In vivo implantation procedure
28. The free DFT® wire ends are then tunneled and externalized at a location on the back between the shoulder blades.
SECTION: Chronic In vivo implantation procedure
29. The free DFT® wire ends and the ground wire are then passed through a custom made, percutaneous silicone plug and soldered to an electrical connector.
SECTION: Chronic In vivo implantation procedure
30. The connector was then fitted inside the silicone tube of the percutaneous plug and the tube was filled with UV curable, medical grade epoxy.
SECTION: Chronic In vivo implantation procedure
31. Blunt dissection is then performed to create a pocket in the back between the skin and muscle.
SECTION: Chronic In vivo implantation procedure
32. The implanted wires and ground wire are placed within this pocket.
SECTION: Chronic In vivo implantation procedure
33. The percutaneous plug’s mesh base was then sutured to the underlying muscle fascia using biodegradable 3-0 sutures.
SECTION: Chronic In vivo implantation procedure
34. All skin incision sites were closed using 4-0 sutures.
SECTION: Chronic autonomic nerve recordings
35.1. The first PCB contains the mating connector to the connector which resides in the implanted percutaneous plug.
SECTION: Chronic autonomic nerve recordings
35.2. The second PCB routes each differential electrode pair to an electrical connector that connects directly to a commercially available 16-amplifier, neural instrumentation amplifier board.
SECTION: Chronic autonomic nerve recordings
36. DFT® wires are soldered to make electrical connections between the two PCB boards for all the CNT yarn electrode implants (up to four total) and the implanted ground wire.
SECTION: Chronic autonomic nerve recordings
37. The electrode connections are routed such that one differential channel is simultaneously recorded with amplifiers 0–7 and the other is captured with amplifiers 8–15.
SECTION: Chronic autonomic nerve recordings
38. The RHD2216 performs the signal filtering, analog signal multiplexing, and analog to digital conversion and is controlled via a digital SPI connection to a commercially available acquisition system (Fig. 1eand Supplementary Fig. 4c).
SECTION: Chronic autonomic nerve recordings
39. Neural signal acquisition was performed and stored to a laptop computer using the following system configurations: 20 k samples/sec/amplifier, DSP offset removal was enabled with the high-pass filter cutoff frequency set at 100 Hz, the amplifier bandpass filters were configured to be between 100 Hz and 9 kHz.
SECTION: Chronic autonomic nerve recordings
40. A manual trigger switch signal is recorded via a separate analog-to-digital input line on the RHD2000 evaluation system.
SECTION: Chronic autonomic nerve recordings
40.1. This trigger signal is synchronized with the recorded neural amplifier data and is also sampled at a rate of 20 k samples/sec.
SECTION: Chronic autonomic nerve recordings
40.2. Holding down a simple push button switch, produces a high logic-level trigger signal which is used to mark different events during the various experiments.
SECTION: Chronic autonomic nerve recordings
40.3. Releasing the push button returns the trigger signal to a low logic-level (Supplementary Fig. 4c).