Protocol for Construction of Rat Nerve Stimulation Cuff Electrodes

Peripheral nerve stimulation has emerged as a platform therapy to treat a wide range of disorders. Continued development and translation of these strategies requires that researchers have access to reliable, customizable electrodes for nerve stimulation. Here, we detail procedures to build three different configurations of cuff electrodes with varying numbers and orientations of contacts for nerve stimulation in rats. These designs are built with simple, widely available materials, using platinum–iridium electrodes assembled into polyurethane tubing. Moreover, the designs can easily be customized to increase versatility and individualize for specific stimulation applications. This protocol provides a resource to facilitate the construction and customization of stimulation cuffs to support preclinical nerve stimulation research.


Introduction
Peripheral nerve stimulation has emerged as a strategy to treat a wide range of neurological disorders. Vagus nerve stimulation (VNS) is one of the most widely used applications and has been used in over 70,000 patients for intractable epilepsy [1]. Recent studies indicate that VNS may be useful for other disorders, including stroke, tinnitus, and PTSD [2][3][4][5][6][7][8][9][10][11][12][13][14][15]. In addition to VNS, other peripheral nerve stimulation therapies include the use of tibial nerve stimulation for bladder control and occipital nerve stimulation for migraines [16,17]. Preclinical development is critical to the effective clinical translation of these emerging therapies. However, broad access to reliable electrodes for nerve stimulation in animal models can limit the ability of researchers to carry out these preclinical studies.
Here, we present a detailed protocol for the micro-construction of different implementations of cuff electrodes for peripheral nerve stimulation to increase access for researchers. The cuffs are similar in design to the standard configurations used in most preclinical studies and are constructed using widely available materials and simple techniques. The use of a microscope aids in increasing standardization across cuffs, quality, and durability. While the cuff electrode design will ultimately depend on the details of each experimental preparation, we describe the construction of a widely-applicable, simple, two-channel circumferential cuff electrode. This cuff design has been employed in a number of studies for vagus nerve stimulation in rats and shares the majority of features with commercially available cuffs from MicroProbes and CorTec [2][3][4][5][6][7]10,[12][13][14][15][18][19][20][21][22][23][24]. Additionally, we provide designs for two additional multichannel cuff electrode configurations which can be modified to suit a greater range of

Sciatic Nerve Stimulation
Rats were anesthetized using ketamine hydrochloride (80 mg/kg, IP injection) and xylazine (10mg/kg, IP). Once the surgical site was shaved, an incision was made on the skin directly above the biceps femoris muscle. The sciatic nerve was exposed by dissecting under the biceps femoris. The gastrocnemius muscle was separated from skin and surrounding tissue using blunt dissection. Cuff electrodes were then placed on the sciatic nerve with leads connected to an isolated programmable stimulator (Model 4100; A-M Systems™; Sequim, WA, USA). The nerve was left in place underneath the gastrocnemius. The cavity was kept full of saline at all times to ensure that the cuff would be operating in a uniform medium with conductance similar to tissue. The Achilles tendon was severed at the ankle and affixed to a force transducer using nylon sutures. The foot was clamped and secured to a stereotaxic frame to prevent movement of the leg during stimulation and to isolate recordings from the gastrocnemius muscle.
Stimulation consisted of 0.5 s trains of biphasic pulses (100 µs pulse width) at 30 Hz with varying current amplitudes delivered using the isolated pulse stimulator. Stimulation intensities were randomly interleaved. Voltage was recorded using a digital oscilloscope (Pico Technology, PicoScope 2204A). The force of muscle contraction was sampled at 10 Hz and recorded through a force transducer which was connected to an analog channel on an Arduino Mega 2560. All components were integrated using MATLAB. Stimulation was delivered every 15 s and each parameter was repeated in triplicate. Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c). Residue removal from the drilling tool bevel using a #11 scalpel blade.

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Use caution when handling either end to prevent injuries. 2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45 • angle and move the needle sideways while rotating it to sharpen the edges (Figure 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c).

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries. 2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c). This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters. 2 Insert the #3 tip at the top of the torch handle. 3 Connect the propane and oxygen tanks to their respective preset regulators. 4 Turn the valves located on the preset regulators to open the oxygen and propane flow.  Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c). This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.

Surface Aid (Optional)
For better handling of the small components we recommend the creation of a small surface aid. Objects such as needles, tape, and the tubing can be easily attached to this surface. 1 Obtain a flat piece of cardboard and fold it over itself to create a small cardboard square or a rectangle. 2 Wrap the entire folded cardboard with masking tape to prevent unfolding. Wrapping the folded cardboard with a total of two or three layers of tape is recommended.

Standard Circumferential Cuff Electrode
A detailed description of the Standard Circumferential Cuff Electrode ( Figure 2) construction process is presented below.

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries. 2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c).

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.  into a sharp blue flame. Opening the valve too fast or too much might cause a loud popping sound. If this occurs, close the oxygen valve, reignite the pilot flame, and repeat this step. CRITICAL STEP Keep the blue flame approximately 1 cm away from the wire when performing Step 1.4. Passing the blue flame too close to the PI wire will result in a wire kink that will damage the wire and irritate the vagus nerve after implantation. 1.4 PAUSE STEP Performing Steps 1.4 and 1.5 will heat the micro-alligator clip and the PI wire.
Always allow approximately 1 min for cool down before touching the clip and the wire after these steps. Looking under the microscope, point the tip of the blue sharp flame to the 1cm PI wire end and pass it all the way across without touching it (approximately 1 cm away from the wire), heating it to a bright orange. Repeat this several times until the wires have fused together which will be indicated by the formation of bubbles in the PI wire (Figure 3a,c). 1.5 Looking under the microscope, touch the end tip of the clamped 1 cm PI wire with the sharp blue flame to melt 1 mm of the PI wire into a ball. We will refer to the small ball and the fused 9 mm of wire collectively as the "small ball end". CRITICAL STEP Close the oxygen and propane valves at the handle of the jewelry torch.
PAUSE STEP Flammable materials required ahead. Previous experience using a jewelry torch is recommended. Use caution working with the torch. Do not ignite the pilot flame pointing at yourself or other individuals.

1.2
Slightly open the propane valve at the torch handle and ignite the pilot flame.

1.3
Slightly open the oxygen valve at the top of the jewelry torch until the pilot flame has turned into a sharp blue flame. Opening the valve too fast or too much might cause a loud popping sound. If this occurs, close the oxygen valve, reignite the pilot flame, and repeat this step.

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries. 2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c). Residue removal from the drilling tool bevel using a #11 scalpel blade.

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters. 1 cm away from the wire), heating it to a bright orange. Repeat this several times until the wires have fused together which will be indicated by the formation of bubbles in the PI wire (Figure 3a,c). 1.5 Looking under the microscope, touch the end tip of the clamped 1 cm PI wire with the sharp blue flame to melt 1 mm of the PI wire into a ball. We will refer to the small ball and the fused 9 mm of wire collectively as the "small ball end". Residue removal from the drilling tool bevel using a #11 scalpel blade.

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Close the oxygen and propane valves at the handle of the jewelry torch. Place the jewelry torch in a secured location until further indication. Release the PI wire from the alligator clip and use a ruler to measure 7.7 cm of PI wire from the melted ball of the small ball end to the spool of PI wire. Then, cut the wire at that location using the surgical scissors. 1. 6 Use a ruler to measure 8 mm of PI wire at the insulated end of the 7.7 cm PI wire (opposite from the small ball end). Secure the wire in the micro-alligator clip. 1.7 Completely melt the 8 mm of PI wire into a ball by re-igniting the jewelry torch and touching the 8 mm PI wire end with the sharp blue flame. We will refer to this melted section as the "big ball end". Completion of Steps 1-1.8 in this section will create a 6.9 cm wire with a big ball end, a 6 cm insulated section (middle portion), and a small ball end (9 mm of fused wire with small ball) ( Figure 3b).
sound. If this occurs, close the oxygen valve, reignite the pilot flame, and repeat this step. CRITICAL STEP Keep the blue flame approximately 1 cm away from the wire when performing Step 1.4. Passing the blue flame too close to the PI wire will result in a wire kink that will damage the wire and irritate the vagus nerve after implantation. 1.4 PAUSE STEP Performing Steps 1.4 and 1.5 will heat the micro-alligator clip and the PI wire.
Always allow approximately 1 min for cool down before touching the clip and the wire after these steps. Looking under the microscope, point the tip of the blue sharp flame to the 1cm PI wire end and pass it all the way across without touching it (approximately 1 cm away from the wire), heating it to a bright orange. Repeat this several times until the wires have fused together which will be indicated by the formation of bubbles in the PI wire (Figure 3a,c). 1.5 Looking under the microscope, touch the end tip of the clamped 1 cm PI wire with the sharp blue flame to melt 1 mm of the PI wire into a ball. We will refer to the small ball and the fused 9 mm of wire collectively as the "small ball end". CRITICAL STEP Close the oxygen and propane valves at the handle of the jewelry torch. Place the jewelry torch in a secured location until further indication. Release the PI wire from the alligator clip and use a ruler to measure 7.7 cm of PI wire from the melted ball of the small ball end to the spool of PI wire. Then, cut the wire at that location using the surgical scissors. 1.6 Use a ruler to measure 8 mm of PI wire at the insulated end of the 7.7 cm PI wire (opposite from the small ball end). Secure the wire in the micro-alligator clip. 1.7 Completely melt the 8 mm of PI wire into a ball by re-igniting the jewelry torch and touching the 8 mm PI wire end with the sharp blue flame. We will refer to this melted section as the "big ball end". Completion of Steps 1-1.8 in this section will create a 6.9 cm wire with a big ball end, a 6 cm insulated section (middle portion), and a small ball end (9 mm of fused wire with small ball) ( Figure 3b).

1.8
Repeat steps 1-1.8 in this section to create a second standard cuff PI wire.

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries.
2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c). Residue removal from the drilling tool bevel using a #11 scalpel blade.

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Steps 1.10-1.16 must be performed under the microscope. 1.9 Insert two gold pins into the female end of the dip socket. 1.10 Use a paper clip to add a miniscule dab of water-soluble flux paste into the mounting holes of the four gold pins (Figure 4a). 1.11 Set the temperature of the soldering iron to 700 • F and allow it to heat (30-60 s) then, touch the top edge of each gold pin for approximately two seconds to melt the flux paste into the mounting holes. Repeat this step a couple more times to make sure the water-soluble flux has covered the entire inside of the gold pins.

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries.
2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c). This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Clean the soldering iron tip as needed using a wet sponge.  (Figure 4b).

Figure 1.
Drilling Tool; (a) 1.5 cm shaft removal from the 22G 1" needle using wire cutters and helping hands. (b) Sharpening of the drilling tool by rotating its flat edge across a nail file in a 45° angle. (c) Residue removal from the drilling tool bevel using a #11 scalpel blade.

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP No solder must drip from the mounting hole. If this occurs, replace that gold pin for a new one and repeat Steps 1.11-1.13. 1.13 Use the tip of the #5 jeweler's forceps to clean any excess solidified flux paste at the top of the gold pins. 1.14 One at a time, touch the top edge of each gold pin with the soldering iron to re-melt the solder inside the mounting holes. At the same time, insert the 3 mm uninsulated end of the 6.9 cm PI wires (one for each gold pin). Then, remove the soldering iron from the gold pins and allow the solder to solidify with the 3 mm uninsulated ends inside the mounting holes ( Figure 4c). This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Keep the wire straight and steady when inserting the big ball end of the PI wires into the mounting holes to prevent any damage to the insulation not being inserted. 1.15 Apply a tiny drop of acrylic (liquid acrylic mixed with powder acrylic) at the top of each gold pin using a fine detail brush ( Figure 4d). This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP No acrylic must drip off the top of the gold pins. If this becomes the case, use a small paper towel to wipe off the acrylic around the gold pin.
1.8 Repeat steps 1-1.8 in this section to create a second standard cuff PI wire.
CRITICAL STEP Steps 1.10-1.16 must be performed under the microscope. 1.9 Insert two gold pins into the female end of the dip socket. 1.10 Use a paper clip to add a miniscule dab of water-soluble flux paste into the mounting holes of the four gold pins (Figure 4a). 1.11 Set the temperature of the soldering iron to 700 °F and allow it to heat (30-60 s) then, touch the top edge of each gold pin for approximately two seconds to melt the flux paste into the mounting holes. Repeat this step a couple more times to make sure the water-soluble flux has covered the entire inside of the gold pins. CRITICAL STEP Clean the soldering iron tip as needed using a wet sponge. 1.12 One at a time, insert the solder wire into the gold pin mounting hole and touch the upper edge of the gold pin to melt the solder. Completely fill the mounting hole with the lead-free solder (Figure 4b).
CRITICAL STEP No solder must drip from the mounting hole. If this occurs, replace that gold pin for a new one and repeat Steps 1.11-1.13. 1.13 Use the tip of the #5 jeweler's forceps to clean any excess solidified flux paste at the top of the gold pins. 1.14 One at a time, touch the top edge of each gold pin with the soldering iron to re-melt the solder inside the mounting holes. At the same time, insert the 3 mm uninsulated end of the 6.9 cm PI wires (one for each gold pin). Then, remove the soldering iron from the gold pins and allow the solder to solidify with the 3 mm uninsulated ends inside the mounting holes ( Figure 4c). CRITICAL STEP Keep the wire straight and steady when inserting the big ball end of the PI wires into the mounting holes to prevent any damage to the insulation not being inserted. 1.15 Apply a tiny drop of acrylic (liquid acrylic mixed with powder acrylic) at the top of each gold pin using a fine detail brush ( Figure 4d). CRITICAL STEP No acrylic must drip off the top of the gold pins. If this becomes the case, use a small paper towel to wipe off the acrylic around the gold pin.   Cuff skeleton (15-20 min). Use a ruler to measure 3 mm of polyurethane tubing and cut it using a single-straight scalpel cut.

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries. 2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c). This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.

CRITICAL STEP
The tube should have no rough edges at either end in order to prevent irritation after implantation. Insert the closed tips of a #5 jeweler's forceps in the 3 mm polyurethane tube, lay them horizontally over your working surface, and place them perpendicular in respect to your shoulders. The side of the 3 mm tube facing upwards will be referred to as the posterior side.  Always allow approximately 1 min for cool down before touching the clip and the wire after these steps. Looking under the microscope, point the tip of the blue sharp flame to the 1cm PI wire end and pass it all the way across without touching it (approximately 1 cm away from the wire), heating it to a bright orange. Repeat this several times until the wires have fused together which will be indicated by the formation of bubbles in the PI wire (Figure 3a,c). 1.5 Looking under the microscope, touch the end tip of the clamped 1 cm PI wire with the sharp blue flame to melt 1 mm of the PI wire into a ball. We will refer to the small ball and the fused 9 mm of wire collectively as the "small ball end". CRITICAL STEP Close the oxygen and propane valves at the handle of the jewelry torch. Place the jewelry torch in a secured location until further indication. Release the PI wire from the alligator clip and use a ruler to measure 7.7 cm of PI wire from the melted ball of the small ball end to the spool of PI wire. Then, cut the wire at that location using the surgical scissors. 1.6 Use a ruler to measure 8 mm of PI wire at the insulated end of the 7.7 cm PI wire (opposite from the small ball end). Secure the wire in the micro-alligator clip. 1.7 Completely melt the 8 mm of PI wire into a ball by re-igniting the jewelry torch and touching the 8 mm PI wire end with the sharp blue flame. We will refer to this melted section as the "big ball end". Completion of Steps 1-1.8 in this section will create a 6.9 cm wire with a big ball end, a 6 cm insulated section (middle portion), and a small ball end (9 mm of fused wire with small ball) ( Figure 3b).
PAUSE STEP Dull forceps are recommended for safety.

2.2
Press and rotate the flat bevel of your drilling tool against the surface of the 3 mm polyurethane tube at a 1.5 mm length (middle of the tube) to carve a hole (Figure 5a). This hole will be referred to as the middle posterior hole.

2.3
Insert a 27G 1 1 /4 " needle through the hub of the drilling tool to remove any tubing stuck on the inside of the shaft. This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.

CRITICAL STEP
Step 2.3 must be performed every time after a hole is drilled. Failure to do this will accumulate polyurethane residues inside the drilling tool, making carving at the surface of the tube extremely difficult.

2.4
Use your drilling tool to drill a hole on the 3 mm polyurethane tube at a 0.5 mm length (1mm below the middle posterior hole). This hole will be referred as the lower posterior hole ( Figure 5b). Sharpen the drilling tool if needed (see "Drilling Tool" in the Equipment Setup section).

2.5
USe your drilling tool to drill a hole at a 2.5 mm length (1 mm above the middle posterior hole). This hole will be referred to as the upper posterior hole (Figure 5c). Completion of this step should create a 3 mm tube with three posterior holes (upper, middle, lower).

2.6
Rotate the #5 forceps 180 • in order to move the posterior side of the tube (with the posterior holes) face-down and the anterior side face-up.

2.7
Use a fine-tip permanent marker to draw a straight line on the surface of the 3 mm tube directly across the three posterior holes (Figure 5d). This mark will be used to indicate a 0 • line on the surface of the tube.

2.8
Use your drilling tool to drill a hole at a 1mm length and 45 • leftwards from the 0 • mark. This hole will be referred to as the left inferior hole and the face-up surface of the 3mm tube will be referred as the anterior side. Sharpen the drilling tool if needed.

2.9
Drill a hole at a 1 mm length and 45 • rightwards from the 0 • mark (mirror from the left inferior hole across the 0 • line) using your drilling tool. This hole will be referred to as the right inferior hole. 2.10 Drill a hole at a 2 mm length and 45 • leftwards from the 0 • mark. (1 mm above the left inferior hole) using your drilling tool. This hole will be referred to as the left superior hole. Sharpen the drilling tool if needed. 2.11 Drill a hole at a 2 mm length and 45 • rightwards from the 0 • mark. (1 mm above the right inferior hole) using your drilling tool. This hole will be referred to as the right superior hole. The right and left inferior and superior holes will be collectively referred to as the lateral holes (Figure 5e).

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries. 2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c). This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Hold the #5 jeweler's forceps inside the tube in a closed position with your hands, so they do not suddenly open when performing Step 2.12. 2.12 Without changing the position of the 3 mm polyurethane tube, use a scalpel to make a straight cut along the entire 0 • mark on the surface of the tube. This cut will be referred to as the anterior cut ( Figure 5f). This is now a completed skeleton. Methods Protoc. 2018, 1, x FOR PEER REVIEW 6 of 29  3.2 Turn the needle 180° and pass it through the right superior hole from the inside to the outside of the skeleton thus forming a small "D" loop between the right superior and the right inferior hole at the inside of the skeleton. 3.3 Make two ligature knots to tie the 3 cm silk end and the long silk end (Supplemental Video).
CRITICAL STEP All ligature knots must be firmly tied to prevent detachment. The ligature knots must rest between the superior and inferior holes. 3.4 Measure 5 cm of silk from the skeleton to the end of the needle and make a cut at that location using the surgical scissors.   Cuff assembly (15-20 min). Thread 30 cm of silk into the eye of the #5 sewing needle.

3.1
Pass the needle through the right inferior hole towards the inside of the skeleton. Leave approximately 3 cm of silk on the outside of the tube.

3
Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c). This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.

CRITICAL STEP
The needle should not go through the left inferior or superior hole at any moment.

3.2
Turn the needle 180 • and pass it through the right superior hole from the inside to the outside of the skeleton thus forming a small "D" loop between the right superior and the right inferior hole at the inside of the skeleton.

3.3
Make two ligature knots to tie the 3 cm silk end and the long silk end (Supplemental Video).
tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries.
2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c).

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP All ligature knots must be firmly tied to prevent detachment. The ligature knots must rest between the superior and inferior holes. 3.4 Measure 5 cm of silk from the skeleton to the end of the needle and make a cut at that location using the surgical scissors.

3.5
Trim the 3 cm silk end to approximately 5 mm. 3.6 Repeat Steps 3.1-3.5 with the left inferior and superior holes. 3.7 Carefully pull each of the 5 cm silk strands in opposite directions to slightly open the inside of the skeleton. Then, use tape strips to attach each of the 5cm silk strands against a flat surface under the microscope (Figure 6a).

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters ( Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries.
2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c).

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters ( Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries. 2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c).

Longitudinal Circumferential Cuff (Figure 7)
1 Gold Pin Wires (10 min). Looking under the microscope, use a ruler to measure 3 mm of platinum-iridium (PI) wire and hold it in the micro-alligator clip. Do not detach or cut the wire from the wire spool. PAUSE STEP Flammable materials required ahead. Previous experience using a jewelry torch is recommended. Do not ignite the pilot flame pointing at yourself or other individuals.

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters ( Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries. 2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c).

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP No portion of the PI wire should go through the right superior or inferior holes during this step. Use the #5 jeweler's forceps to grab the small ball at the gold pin wire small ball end and loop it around the left superior hole once (from the inside to the outside and to the inside again). Insulated wire should wrap the superior hole, but only uninsulated wire should re-enter the inside of the tube. Figure 1. Drilling Tool; (a) 1.5 cm shaft removal from the 22G 1" needle using wire cutters and helping hands. (b) Sharpening of the drilling tool by rotating its flat edge across a nail file in a 45° angle. (c) Residue removal from the drilling tool bevel using a #11 scalpel blade.

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.

CRITICAL STEP
The edge between the uninsulated and the insulated sections of the gold pin wire should rest at the left superior hole. 3.9 Tighten the loop around the left superior hole by pulling both ends of the gold pin wire in opposite directions. 3.10 Use the #5 jeweler's forceps to pass the remaining portion of the small ball end through the right superior hole (from the inside to the outside of the skeleton) ( Figure 6b). 3.11 Insert a pipette tip through the anterior cut (parallel to the tube) to push the uninsulated wire inside the skeleton against the inner wall of the tube.

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Once the gold pin wiring has been completed, make sure the uninsulated sections of the wire inside the skeleton are not damaged (broken, fried, dented, etc.). If damaged, remove the entire gold pin wire and replace it with a new one.

3
Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c). polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters ( Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries.
2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c).

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries. 2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c). This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Similarly to Step 3.16, the "D" loop formed between the lower posterior hole and the middle posterior hole should pull the uninsulated melted section of the gold pin wire between these holes against the inner wall of the tube.
3.19 Make three ligature knots using the 3 cm silk end and the silk with the needle end on the outside of the skeleton (posterior side). Then, remove the tape from the 5 cm silk strands to release the skeleton. 3.20 Use the fine-tip acrylic brush to add a miniscule drop of wet acrylic to each of the lateral holes and the lateral ligature knots. Wait 5 min for the acrylic to dry before touching the now completed VNS cuff. Residue removal from the drilling tool bevel using a #11 scalpel blade.

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Make sure no acrylic drips inside of the skeleton. Clean the inside of the skeleton immediately with a surgical spear (Weck-Cell) wet in 70% isopropyl rubbing alcohol if this becomes the case. This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Failure to maintain an acrylic-free environment inside the skeleton could affect the reliability of stimulation by electrode-nerve contact obstruction.
This is now a completed Standard VNS cuff (Figure 6f).   Always allow approximately 1 min for cool down before touching the clip and the wire after these steps.

Longitudinal Circumferential Cuff
Looking under the microscope, point the tip of the blue sharp flame to the 1cm PI wire end and pass it all the way across without touching it (approximately 1 cm away from the wire), heating it to a bright orange. Repeat this several times until the wires have fused together which will be indicated by the formation of bubbles in the PI wire (Figure 3a,c). 1.5 Looking under the microscope, touch the end tip of the clamped 1 cm PI wire with the sharp blue flame to melt 1 mm of the PI wire into a ball. We will refer to the small ball and the fused 9 mm of wire collectively as the "small ball end". CRITICAL STEP Close the oxygen and propane valves at the handle of the jewelry torch. Place the jewelry torch in a secured location until further indication. Release the PI wire from the alligator clip and use a ruler to measure 7.7 cm of PI wire from the melted ball of the small ball end to the spool of PI wire. Then, cut the wire at that location using the surgical scissors. 1.6 Use a ruler to measure 8 mm of PI wire at the insulated end of the 7.7 cm PI wire (opposite from the small ball end). Secure the wire in the micro-alligator clip. 1.7 Completely melt the 8 mm of PI wire into a ball by re-igniting the jewelry torch and touching the 8 mm PI wire end with the sharp blue flame. We will refer to this melted section as the "big ball end". Completion of Steps 1-1.8 in this section will create a 6.9 cm wire with a big ball end, a 6 cm insulated section (middle portion), and a small ball end (9 mm of fused wire with small ball) (Figure 3b).

PAUSE STEP Performing
Step 1.3 may dangerously heat the micro-alligator clip and the PI wire. Always allow approximately 1 minute for cool down before touching the clip and the wire after these steps.

1.3
Briefly touch the 3 mm end of the PI wire with the pilot flame to burn off the insulation covering the PI wires. We will refer to this uninsulated portion as the 3 mm uninsulated end. 1.4 Slightly open the oxygen valve at the top of the jewelry torch until the pilot flame has turned into a sharp blue flame. Opening the valve too fast or too much might cause a loud popping sound. If this occurs, close the oxygen valve, reignite the pilot flame, and repeat this step. 1.5 Looking under the microscope, touch the end tip of the clamped 3 mm PI wire with the sharp blue flame to completely melt it into a ball. We will refer to it as the "big ball end" (Figure 8a). This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Close the oxygen valve at the handle of the jewelry torch and place the jewelry torch in a secured location until further indication.   Cuff skeleton (5 min). Under the microscope, use a ruler to measure 4 mm of polyurethane tubing and cut it in a single straight scalpel cut.

1.6
Release the big ball end from the alligator clip and use a ruler to measure 7.6 cm of PI wire starting from the big ball end. Then, cut the wire at that location using the surgical scissors. 1.7 Use a ruler to measure 6 mm of PI wire at the insulated end of the 7.6 cm PI wire (opposite from the big ball end) and clamp it with the micro-alligator clip. 1.8 Burn off the insulation of the 6 mm clamped PI wire by briefly touching it with the pilot flame. We will refer to this uninsulated section as the 6 mm uninsulated end. 1.9 Once again, slightly open the oxygen valve at the jewelry torch to turn the pilot flame into the sharp blue flame and melt 1 mm of PI wire at the tip of the clamped 6 mm PI wire. We will refer to the uninsulated 5 mm of PI wire and the 1mm melted ball collectively as the "small ball end" (Figure 8b). Completion of Steps 1-1.9 in this section will create a 7.5 cm wire that includes: a big ball end, a 6.7 cm insulated section (middle portion), and a small ball end. 1.10 Repeat Steps 1-1.9 three more times to create a total of four longitudinal-cuff PI wires.

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Responsibly and safely disarm the jewelry torch set up after completion (make sure all valves are closed before dismantling).    Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel (Figure 1c).  Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries.
2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel (Figure 1c).  Cuff skeleton (5 min). Under the microscope, use a ruler to measure 4 mm of polyurethane tubing and cut it in a single straight scalpel cut.

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries.
2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel (Figure 1c).

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries.
2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel (Figure 1c).  This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.

CRITICAL STEP
The 4 mm polyurethane tube should be firmly stuck to the forceps. Make a single straight scalpel cut along the length of the tube at the face-up surface. We will refer to this as the anterior cut (Figure 9b).

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Hold the #5 forceps tightly closed when making the cut to prevent a sudden opening of the forceps.

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Use the straight indentation between the tips of the #5 forceps to guide the scalpel across the surface of the tube.

2.3
Use a fine-tip permanent marker to draw four points. The first mark will be located at one of the flat edges of the tube at a 0 mm length, and 45 • to the right of the anterior cut. The remaining 3 marks should be drawn at the same flat edge 90 • apart in a clockwise direction. We will refer to these as Marks B, D, F, and H respectively.

2.4
Use the same marker to draw four more points at the opposite flat edge of the tube. These marks should be at reflected positions from Marks B, D, F, and H. We will refer to these marks as Marks A, C, E, and G ( Figure 9c).  CRITICAL STEP The tube should have no rough edges at either end in order to prevent irritation in the animal after implantation.
CRITICAL STEP This tube will be defined to be 4mm in length and a 360° surface (Figure 9a). 2.1 Pass the closed tips of a #5 jeweler's forceps through the inside of the 4mm polyurethane tube.
PAUSE STEP Dull forceps are recommended to prevent any possible stabbing injuries. 2.2 Lay the forceps with the polyurethane tube flat against your working surface. Then, orientate the tube perpendicular to your shoulders. CRITICAL STEP The 4 mm polyurethane tube should be firmly stuck to the forceps. Make a single straight scalpel cut along the length of the tube at the face-up surface. We will refer to this as the anterior cut (Figure 9b).
CRITICAL STEP Hold the #5 forceps tightly closed when making the cut to prevent a sudden opening of the forceps.
CRITICAL STEP Use the straight indentation between the tips of the #5 forceps to guide the scalpel across the surface of the tube. 2.3 Use a fine-tip permanent marker to draw four points. The first mark will be located at one of the flat edges of the tube at a 0 mm length, and 45° to the right of the anterior cut. The remaining 3 marks should be drawn at the same flat edge 90° apart in a clockwise direction. We will refer to these as Marks B, D, F, and H respectively. 2.4 Use the same marker to draw four more points at the opposite flat edge of the tube. These marks should be at reflected positions from Marks B, D, F, and H. We will refer to these marks as Marks A, C, E, and G ( Figure 9c). CRITICAL STEP Mark A must be at the opposite edge of the tube from Mark B, Mark C is opposite to Mark D, Mark E is opposite to Mark F, and Mark G is opposite to Mark H.
CRITICAL STEP Completing the cuff skeleton section will result in a 4mm tube containing an anterior cut and a total of eight marks (four at each flat end of the tube) (Figure 9c). 3 Cuff assembly (20 min). Thread 30 cm of silk into the eye of the #5 sewing needle. 3.1 Pierce the exterior surface of the tube with the silk-threaded needle 30° to the right of the anterior cut at a 1 mm length. 3 Cuff assembly (20 min). Thread 30 cm of silk into the eye of the #5 sewing needle.

3.1
Pierce the exterior surface of the tube with the silk-threaded needle 30 • to the right of the anterior cut at a 1 mm length.

3.2
Pass the entire needle through the puncture (and through the anterior cut) pulling the silk until approximately 3 cm is left on outside the tube. We will refer to this as the short silk end.

3.3
Turn the needle around, pass it through the anterior cut, and pierce the inside surface of the tube 30 • to the right of the anterior cut 2 mm above the puncture made in the previous step. 3 .4 Pass the entire needle through the puncture made in the previous step (towards the outside of the tube) until the silk between the two punctures has formed a "D" loop against the inside surface of the tube. The silk exiting the tube (with the needle at the tip) will be referred to as the long silk end. 3.5 Make two ligature knots at the outside of the skeleton using the short silk end and the long silk end. See supplemental material for a detailed description of the ligature knotting technique.

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP All ligature knots must be firmly tied to prevent detachment during cuff assembly and after surgical implantation.

3.6
Use a ruler to measure 6 cm of silk from the ligature knot towards the needle and make a cut with the surgical scissors at that location.

3.7
Trim the 3 cm end to approximately 2 mm using the surgical scissors.

3.8
Apply a miniscule drop of UV glue to the ligature knot with the eye of a clean #5 sewing needle.

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.

CRITICAL STEP
The UV glue drop must be just big enough to cover the knot. Passing the blue flame too close to the PI wire will result in a wire kink that will damage the wire and irritate the vagus nerve after implantation. 1.4 PAUSE STEP Performing Steps 1.4 and 1.5 will heat the micro-alligator clip and the PI wire.
Always allow approximately 1 min for cool down before touching the clip and the wire after these steps. Looking under the microscope, point the tip of the blue sharp flame to the 1cm PI wire end and pass it all the way across without touching it (approximately 1 cm away from the wire), heating it to a bright orange. Repeat this several times until the wires have fused together which will be indicated by the formation of bubbles in the PI wire (Figure 3a,c). 1.5 Looking under the microscope, touch the end tip of the clamped 1 cm PI wire with the sharp blue flame to melt 1 mm of the PI wire into a ball. We will refer to the small ball and the fused 9 mm of wire collectively as the "small ball end". CRITICAL STEP Close the oxygen and propane valves at the handle of the jewelry torch. Place the jewelry torch in a secured location until further indication. Release the PI wire from the alligator clip and use a ruler to measure 7.7 cm of PI wire from the melted ball of the small ball end to the spool of PI wire. Then, cut the wire at that location using the surgical scissors. 1.6 Use a ruler to measure 8 mm of PI wire at the insulated end of the 7.7 cm PI wire (opposite from the small ball end). Secure the wire in the micro-alligator clip. 1.7 Completely melt the 8 mm of PI wire into a ball by re-igniting the jewelry torch and touching the 8 mm PI wire end with the sharp blue flame. We will refer to this melted section as the "big ball end". Completion of Steps 1-1.8 in this section will create a 6.9 cm wire with a big ball end, a 6 cm insulated section (middle portion), and a small ball end (9 mm of fused wire with small ball) (Figure 3b).
PAUSE STEP Put UV goggles on. UV goggles must be worn at all times as eye protection when using UV light.

3.9
Cure the UV glue covering the ligature knot by shining UV light over it for 10 s. 3.10 Use a tape strip to attach the 6cm silk strand to the flat working surface under the microscope. 3.11 Repeat Steps 3.1 through 3.10 with the threaded needle at the left side of the anterior cut (instead of the right side as indicated by these steps). This will result in a total of two 6 cm silk strands connecting to their respective ligature knots (one on each side of the anterior cut) covered in solid UV glue taped to the working surface ( Figure 10a).
container. CRITICAL STEP Use caution when handling either end to prevent injuries.
2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel (Figure 1c).  Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries.
2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel (Figure 1c).

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.

CRITICAL STEP
The inside surface of the skeleton should be slightly visible through the anterior cut. If this is not the case, remove the tape covering one of the 6 cm silk strands, pull the silk, and reapply the tape. 3.12 Use a clean #5 needle to pierce the exterior surface of the tube 0.5 mm directly above Mark A. Move 3 4 of the needle into the tube (and through the anterior cut) leaving the last quarter on the outside of the initial puncture (Figure 10b).

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries.
2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel (Figure 1c).

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Use your #5 forceps to press against the inner wall of the tube at that location to make the perforation easier. 3.13 Pass the uninsulated end of a gold pin wire through the eye of the #5 needle piercing the tube (until half of the wire has gone through).

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries.
2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c).  Residue removal from the drilling tool bevel using a #11 scalpel blade.

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.

CRITICAL STEP
The gold pin end of the gold pin wire should remain outside of the tube at all times.

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Continue pulling until the gold pin wire has naturally slipped out of the eye of the needle.

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters. Always allow approximately 1 min for cool down before touching the clip and the wire after these steps. Looking under the microscope, point the tip of the blue sharp flame to the 1cm PI wire end and pass it all the way across without touching it (approximately 1 cm away from the wire), heating it to a bright orange. Repeat this several times until the wires have fused together which will be indicated by the formation of bubbles in the PI wire (Figure 3a,c). 1.5 Looking under the microscope, touch the end tip of the clamped 1 cm PI wire with the sharp blue flame to melt 1 mm of the PI wire into a ball. We will refer to the small ball and the fused 9 mm of wire collectively as the "small ball end". CRITICAL STEP Close the oxygen and propane valves at the handle of the jewelry torch. Place the jewelry torch in a secured location until further indication. Release the PI wire from the alligator clip and use a ruler to measure 7.7 cm of PI wire from the melted ball of the small ball end to the spool of PI wire. Then, cut the wire at that location using the surgical scissors. 1.6 Use a ruler to measure 8 mm of PI wire at the insulated end of the 7.7 cm PI wire (opposite from the small ball end). Secure the wire in the micro-alligator clip. 1.7 Completely melt the 8 mm of PI wire into a ball by re-igniting the jewelry torch and touching the 8 mm PI wire end with the sharp blue flame. We will refer to this melted section as the "big ball end". Completion of Steps 1-1.8 in this section will create a 6.9 cm wire with a big ball end, a 6 cm insulated section (middle portion), and a small ball end (9 mm of fused wire with small ball) (Figure 3b).

CRITICAL STEP
PAUSE STEP Put UV goggles on. 3.19 Apply a small drop of UV glue to the puncture below Mark B using the needle in Step 3.8 to cover the small ball end of the gold pin wire exiting the tube. Then, cure the UV glue for ten seconds with the UV light. 3.20 Repeat steps 3.11-3.19 with the remaining marks. Notice that Marks D, F, and H will be used instead of Mark B. Similarly, Marks C, E, and G will be used instead of Mark A (Figure 10c).
needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel (Figure 1c).

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.

CRITICAL STEP
The inside of the tube should now contain four 3 mm uninsulated leads 90 • apart running across the length of the tube.

4
Cuff Knotting (10-15 min). Use the silk-threaded needle to pierce the exterior surface of the tube (and through the anterior cut) 15 • to the right of the anterior cut at a 2 mm length. Continue pulling the needle until approximately 3 cm of silk is left on the outside. We will refer to the 3 cm silk piece as the short silk end.

4.1
Turn with the needle 180 • , pass it through the anterior cut, and pierce the inside surface medially, between the uninsulated A-B and C-D leads. Continue pulling the needle towards the outside of the tube until a "D" loop has formed at the inside of the tube.

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries. 2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel (Figure 1c).

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.

CRITICAL STEP
The "D" loop should press the uninsulated lead running from Mark A to Mark B against the inside surface of the tube.

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries. 2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel (Figure 1c).

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.

CRITICAL STEP
The long piece of silk going towards the outside of the tube (with the needle at the end) will be referred to as the long silk end.

4.2
Make a ligature knot at the outside of the tube using the short and long silk ends.

4.3
Use the scissors to trim both ends of the ligature knot to approximately 3 mm. Apply a small drop of UV glue to the ligature knot with the eye of the needle and cure it by shining UV light over the glue for ten seconds. The UV glue drop should be small enough to only cover the knot, leaving the silk punctures to the right and left of the knot uncovered (preferably).

4.5
Repeat Steps to 4-4.4 to create three more "D" loops (each pressing a lead against the inside surface of the tube) and three more ligature knots. However, instead of piercing at the location indicated in Step 4, use the last puncture of the previous knot to move the needle from the outside to the inside (and through the anterior cut) of the tube.

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP It will only be necessary to make new punctures when moving the needle from the inside to the outside of the tube. 4.6 One at a time, pull the insulated section of each gold pin wire above the gold pin to straighten the longitudinal uninsulated leads inside the tube, apply a small drop of UV glue at the B, D, F, and H punctures, and cure them under UV light for 15 s (Figure 10d).
Methods Protoc. 2018, 1, x FOR PEER REVIEW 13 of 29 CRITICAL STEP A 3 mm uninsulated PI wire "D" loop should be formed against the inner wall of the tube between the punctures made between Mark A and B. 3.17 Hold the small ball at the small ball end of the gold pin with the #5 forceps and the insulated portion of the PI wire next to the gold pin with your fingertips. Then, pull them in opposite directions to flatten the "D" loop at the inside of the tube. 3.18 While keeping the "D" loop flat, retreat the gold pin wire through the punctures by pulling the gold pin end of the gold pin wire. Stop as soon as the last bit of the insulated section becomes aligned with the puncture above Mark A. PAUSE STEP Put UV goggles on. 3.19 Apply a small drop of UV glue to the puncture below Mark B using the needle in Step 3.8 to cover the small ball end of the gold pin wire exiting the tube. Then, cure the UV glue for ten seconds with the UV light. 3.20 Repeat steps 3.11-3.19 with the remaining marks. Notice that Marks D, F, and H will be used instead of Mark B. Similarly, Marks C, E, and G will be used instead of Mark A (Figure 10c). CRITICAL STEP The inside of the tube should now contain four 3 mm uninsulated leads 90° apart running across the length of the tube.  Cuff Knotting (10-15 min). Use the silk-threaded needle to pierce the exterior surface of the tube (and through the anterior cut) 15° to the right of the anterior cut at a 2 mm length. Continue pulling the needle until approximately 3 cm of silk is left on the outside. We will refer to the 3 cm silk piece as the short silk end.

Variable Multi-Contact Circumferential Cuff
A detailed description of the Variable Multi-Contact Circumferential Cuff Electrode ( Figure 11) construction process is presented below.

5
Cuff skeleton (10-15 min). Use a ruler to measure 6mm of polyurethane tubing and cut it using a scalpel. We will treat this tube as a cylinder of 6mm in length. Passing the blue flame too close to the PI wire will result in a wire kink that will damage the wire and irritate the vagus nerve after implantation. 1.4 PAUSE STEP Performing Steps 1.4 and 1.5 will heat the micro-alligator clip and the PI wire.
Always allow approximately 1 min for cool down before touching the clip and the wire after these steps. Looking under the microscope, point the tip of the blue sharp flame to the 1cm PI wire end and pass it all the way across without touching it (approximately 1 cm away from the wire), heating it to a bright orange. Repeat this several times until the wires have fused together which will be indicated by the formation of bubbles in the PI wire (Figure 3a,c). 1.5 Looking under the microscope, touch the end tip of the clamped 1 cm PI wire with the sharp blue flame to melt 1 mm of the PI wire into a ball. We will refer to the small ball and the fused 9 mm of wire collectively as the "small ball end". CRITICAL STEP Close the oxygen and propane valves at the handle of the jewelry torch. Place the jewelry torch in a secured location until further indication. Release the PI wire from PAUSE STEP Flammable materials required ahead. Previous experience using a jewelry torch is recommended. Do not ignite the pilot flame pointing at yourself or other individuals. Always allow approximately 1 min for cool down before touching the clip and the wire after these steps. Looking under the microscope, point the tip of the blue sharp flame to the 1cm PI wire end and pass it all the way across without touching it (approximately 1 cm away from the wire), heating it to a bright orange. Repeat this several times until the wires have fused together which will be indicated by the formation of bubbles in the PI wire (Figure 3a,c).

PAUSE STEP Performing
Step 1.2 may dangerously heat the micro-alligator clip and the PI wire. Always allow approximately 1 minute for cool down before touching the clip and the wire after these steps.

1.2
Briefly touch the 3 mm end of the PI wire with the pilot flame to burn off the insulation covering the PI wires. We will refer to this uninsulated portion as the 1 mm uninsulated end.

1.3
Slightly open the oxygen valve at the top of the jewelry torch until the pilot flame has turned into a sharp blue flame. Opening the valve too fast or too much might cause a loud popping sound. If this occurs, close the oxygen valve, reignite the pilot flame, and repeat this step.

1.4
Melt 1 mm of PI wire at the tip of the clamped 3 mm PI wire by touching it with the sharp blue flame. We will refer to the uninsulated 2 mm of PI wire and the 1mm melted ball collectively as the "small ball end" (Figure 12a).

1.5
Release the small ball end of the 60 • PI wire from the alligator clip and use a ruler to measure 7.5 cm of PI wire from the tip of the 2 mm uninsulated end to the spool of PI wire. Then, cut the wire at that location using the surgical scissors. 1. 6 USe a ruler to measure 3 mm of PI wire at the insulated end of the 7.5 cm PI wire (opposite from the small ball end). Then, hold it in the micro-alligator clip. 1.7 Completely melt the 3 mm locked PI wire by touching it with the sharp blue flame. We will refer to this end as the big ball end (Figure 12d). Completion of steps 1-1.7 in this section will create a 7.2 cm wire with a 2 mm uninsulated end (small ball end), a 7 cm insulated section (middle portion), and a big ball end. 1.8 Repeat Steps 1-1.7 to create a second 7.2 cm PI wire. Always allow approximately 1 min for cool down before touching the clip and the wire after these steps. Looking under the microscope, point the tip of the blue sharp flame to the 1cm PI wire end and pass it all the way across without touching it (approximately 1 cm away from the wire), heating it to a bright orange. Repeat this several times until the wires have fused together which will be indicated by the formation of bubbles in the PI wire (Figure 3a,c). 1.5 Looking under the microscope, touch the end tip of the clamped 1 cm PI wire with the sharp blue flame to melt 1 mm of the PI wire into a ball. We will refer to the small ball and the fused 9 mm of wire collectively as the "small ball end". CRITICAL STEP Close the oxygen and propane valves at the handle of the jewelry torch. Place the jewelry torch in a secured location until further indication. Release the PI wire from the alligator clip and use a ruler to measure 7.7 cm of PI wire from the melted ball of the small ball end to the spool of PI wire. Then, cut the wire at that location using the surgical scissors. 1.6 Use a ruler to measure 8 mm of PI wire at the insulated end of the 7.7 cm PI wire (opposite from the small ball end). Secure the wire in the micro-alligator clip. 1.7 Completely melt the 8 mm of PI wire into a ball by re-igniting the jewelry torch and touching the 8 mm PI wire end with the sharp blue flame. We will refer to this melted section as the "big ball end". Completion of Steps 1-1.8 in this section will create a 6.9 cm wire with a big ball end, a 6 cm insulated section (middle portion), and a small ball end (9 mm of fused wire with small ball) (Figure 3b).

PAUSE STEP Performing
Step 2.1 may dangerously heat the micro-alligator clip and the PI wire. Always allow approximately 1 min for cool down before touching the clip and the wire after these steps.

2.1
Briefly touch the 5mm end of the PI wire with the pilot flame to burn off the insulation covering the PI wires. We will refer to this uninsulated portion as the 5 mm uninsulated end.

2.2
Slightly open the oxygen valve at the top of the jewelry torch until the pilot flame has turned into a sharp blue flame. Opening the valve too fast or too much might cause a loud popping sound. If this occurs, close the oxygen valve, reignite the pilot flame, and repeat this step.

2.3
Melt 1 mm of PI wire at the tip of the clamped 5 mm PI wire by touching it with the sharp blue flame. We will refer to the uninsulated 4mm of PI wire and the 1 mm melted ball collectively as the "small ball end" (Figure 12b).

2.4
Release the small ball end of the 120 • PI wire from the alligator clip and use a ruler to measure 7.7 cm of PI wire from the tip of the small ball end to the spool of PI wire. Then, cut the wire at that location using the surgical scissors.

2.5
Use a ruler to measure 3 mm of PI wire at the insulated end of the 7.7 cm PI wire (opposite from the small ball end). Then, hold it in the micro-alligator clip.

2.6
Completely melt the 3 mm locked PI wire by touching it with the sharp blue flame. We will refer to this end as the big ball end (Figure 12d). Completion of Steps 2-2.6 in this section will create a 7.4 cm wire with a 4 mm uninsulated end (small ball end), a 7 cm insulated section (middle portion), and a big ball end.

2.7
Repeat Steps 2-2.6 to create a second 7.4 cm PI wire. 3.1 PAUSE STEP Performing Step 3.1 may dangerously heat the micro-alligator clip and the PI wire. Always allow approximately 1 minute for cool down before touching the clip and the wire after these steps. Briefly touch the 8 mm end of the PI wire with the pilot flame to burn off the insulation covering the PI wires. We will refer to this uninsulated portion as the 8 mm uninsulated end.

3.2
Slightly open the oxygen valve at the top of the jewelry torch until the pilot flame has turned into a sharp blue flame. Opening the valve too fast or too much might cause a loud popping sound. If this occurs, close the oxygen valve, reignite the pilot flame, and repeat this step. 3.3 Melt 1 mm of PI wire at the tip of the clamped 8 mm PI wire by touching it with the sharp blue flame. We will refer to the uninsulated 7 mm of PI wire and the 1 mm melted ball collectively as the "small ball end" of the 270 • PI wire (Figure 12c).

3.4
Release the PI wire small ball end from the alligator clip and use a ruler to measure 8 cm of PI wire from the small ball end to the spool of PI wire. Then, cut the wire at that location using the surgical scissors. 3.5 Use a ruler to measure 3 mm of PI wire at the insulated end of the 8 cm PI wire (opposite from the small ball end). Then, hold it in the micro-alligator clip. 3.6 Completely melt the 3 mm locked PI wire by touching it with the sharp blue flame. We will refer to this end as the big ball end (Figure 12d). Completion of Steps 3-3.6 in this section will create a 7.7 cm wire with a 7 mm uninsulated end (small ball end), a 7 cm insulated section (middle portion), and a big ball end.

3.7
Repeat Steps 3-3.6 to create a second 7.7 cm PI wire.

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.

CRITICAL STEP
Steps 4-4.7 must be performed under the microscope. Use a paper clip to add a miniscule dab of water-soluble flux paste into the mounting holes of the six gold pins (Figure 12e).

4.3
Set the temperature of the soldering iron to 700 • F and touch the top edge of each gold pin for approximately two seconds to melt the flux paste into the mounting holes. Repeat this step a couple more times to make sure the water-soluble flux has covered the entire inside of the gold pins.
CRITICAL STEP Use caution when handling either end to prevent injuries.
2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel (Figure 1c).

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Clean the soldering iron tip as needed using a wet sponge. 4.4 One at a time, use the soldering iron to completely fill the mounting hole of each gold pin with lead-free solder.

1
Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries.
2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel (Figure 1c). One at a time, touch the top edge of each gold pin with the soldering iron to re-melt the solder inside the mounting holes. At the same time, insert the big ball end of the PI wire (one for each gold pin). Then, remove the soldering iron from the gold pin and allow the solder to solidify with the big ball end inside the mounting hole (Figure 12f).

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries.
2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel (Figure 1c).

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries. 2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c).  This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP All procedures in this section must be performed under a microscope.

5
Cuff skeleton (10-15 min). Use a ruler to measure 6mm of polyurethane tubing and cut it using a scalpel. We will treat this tube as a cylinder of 6mm in length.

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Make a single straight cut when performing this step. The tube should have no rough edges at either end in order to prevent irritation in the animal.

5.1
Insert the tip of one 1 3 4 " paper clip into the 6mm polyurethane tube and lay them horizontally over your working surface. Orient the polyurethane tube perpendicular to your shoulders. We will refer to the current face-up surface of the 6mm tube as the posterior side.

5.2
USe a fine-tip permanent marker to draw six points along the length of the 6 mm tube. The first point should be drawn 0.5 mm above the bottom edge of the tube and will be referred to as Point 1. The remaining points should be drawn 1 mm apart in a straight line towards the top edge of the tube and referred to as Points 2-6 ( Figure 13a).

5.3
Move the posterior side of the tube face-down by rotating the 6 mm polyurethane tube 180 • at the tip of the paper clip. Then, use a fine-tip marker and a ruler to draw a straight line along the entire length of the tube directly across from Points 1-6. We will refer to this as the 0 • line (Figure 13b).

5.4
USe the permanent marker and the ruler to draw a point at a 2.

5
Cuff skeleton (10-15 min). Use a ruler to measure 6mm of polyurethane tubing and cut it using a scalpel. We will treat this tube as a cylinder of 6mm in length. Use the permanent fine-tip marker to indicate two points 0.46 mm to the right and left of the point drawn at a 2.5 mm length. Use the space between the centimeter line and the adjacent millimeter marks of the ruler to approximate the correct location. A little less than half way between the centimeter mark and the adjacent millimeter marks of the ruler may be used as a visual approximation. Use a caliper to verify your placement once the points have been drawn. We will refer to the two marks as the lower right and left 270° lead marks.  USe the permanent fine-tip marker to indicate two points 0.46 mm to the right and left of the point drawn at a 2.5 mm length. Use the space between the centimeter line and the adjacent millimeter marks of the ruler to approximate the correct location. A little less than half way between the centimeter mark and the adjacent millimeter marks of the ruler may be used as a visual approximation. Use a caliper to verify your placement once the points have been drawn. We will refer to the two marks as the lower right and left 270 • lead marks. line and the adjacent millimeter marks of the ruler to visually approximate the correct location and use a caliper to verify the placement. We will refer to these points as the lower 60 • lead marks. 5.11 Repeat Steps 5.10 and 5.11 for Point 4. We will refer to the new marks as the upper 60 • lead marks. 5.12 Place the ruler over Point 2 and align it with a centimeter mark. The ruler should cover only half the point when placed over. 5.13 Use the fine-tip marker to indicate two points 0.61 mm to the right and left of the Point 2. Use the space between the centimeter line and the adjacent millimeter marks of the ruler to visually approximate the correct location. We will refer to the two marks as the lower right and left 120 • lead marks. 5.14 Repeat Steps 5.13 and 5.14 over Point 5, located at a 4.5 mm length from the bottom edge of the tube. We will refer to the two marks sideways from Point 5 as the upper right and left 120 • lead marks. 5.15 Turn the tube 180 • on the paper clip to move the anterior side of the tube face-up. Then, make a single-straight scalpel cut along the entire 0 • line of the tube. We will refer to this as the anterior cut. 6 Cuff assembly (20-30 min). Thread 30 cm of silk into the eye of the #5 sewing needle.

6.1
Pierce the exterior surface of the tube with the silk-threaded needle 90 • to the right of the anterior cut and at a 5 mm tube length from the bottom edge of the tube.

6.2
Pass the entire needle through the pierce and pull the silk until approximately 3 cm are left on outside the tube. We will refer to this as the short silk end. 6.3 Turn the needle around, pass it through the anterior cut, and pierce the inside surface of the tube 90 • to the right of the anterior cut 1 mm below the previous puncture (Step 6.1). Pass the entire needle through the puncture and pull the needle until the silk between the two pierces has formed a "D" loop against the inside surface of the tube. The silk exiting the tube (with the needle at the tip) will be referred to as the long silk end. 6.4 Make two ligature knots at the outside of the skeleton using the short silk end and the long silk end. See supplemental material for a detailed description of the ligature knotting technique. This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP All ligature knots must be firmly tied to prevent detachment during cuff assembly and after surgical implantation. 6.5 Use a ruler to measure 6 cm of silk from the ligature knot towards the needle and make a cut using the surgical scissors at that location. 6.6 Trim the 3 cm end to approximately 2 mm using the surgical scissors. Always allow approximately 1 min for cool down before touching the clip and the wire after these steps. Looking under the microscope, point the tip of the blue sharp flame to the 1cm PI wire end and pass it all the way across without touching it (approximately 1 cm away from the wire), heating it to a bright orange. Repeat this several times until the wires have fused together which will be indicated by the formation of bubbles in the PI wire (Figure 3a,c). 1.5 Looking under the microscope, touch the end tip of the clamped 1 cm PI wire with the sharp blue flame to melt 1 mm of the PI wire into a ball. We will refer to the small ball and the fused 9 mm of wire collectively as the "small ball end". CRITICAL STEP Close the oxygen and propane valves at the handle of the jewelry torch. Place the jewelry torch in a secured location until further indication. Release the PI wire from the alligator clip and use a ruler to measure 7.7 cm of PI wire from the melted ball of the small ball end to the spool of PI wire. Then, cut the wire at that location using the surgical scissors. 1.6 Use a ruler to measure 8 mm of PI wire at the insulated end of the 7.7 cm PI wire (opposite from the small ball end). Secure the wire in the micro-alligator clip. 1.7 Completely melt the 8 mm of PI wire into a ball by re-igniting the jewelry torch and touching the 8 mm PI wire end with the sharp blue flame. We will refer to this melted section as the "big ball end". Completion of Steps 1-1.8 in this section will create a 6.9 cm wire with a big ball end, a 6 cm insulated section (middle portion), and a small ball end (9 mm of fused wire with small ball) (Figure 3b).

PAUSE STEP
Put UV goggles on. UV goggles must be worn at all times as eye protection when using UV light. 6.7 Apply a miniscule drop of UV glue to the ligature knot with the eye of a clean #5 sewing needle. This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.

CRITICAL STEP
The UV glue drop must be just big enough to cover the knot. 6.8 Cure the UV glue covering the ligature knot by shining UV light over it for 10 s. 6.9 Use a tape strip to attach the 6 cm silk strand to the flat working surface under the microscope perpendicular to the tube. 6.10 Repeat Steps 6.2-6.9 with the threaded needle 90 • to the right of the anterior cut. The D loop should start at a 2 mm length and exit the tube at a 1 mm length. 6.11 Repeat Steps 6.2-6.10 at the left side of the tube. Use the same measurements to create mirror ligature knots on the left side of the tube.
needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel (Figure 1c). This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Completion of these steps will result in a total of four ligature knots with two on each side of the tube. (Figure 14a). 6.12 Pass the uninsulated end of one of the 270 • gold pin wires through the eye of a #5 sewing needle.

1
Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries.
2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel (Figure 1c). This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Do not loop or knot the wire to the needle at any point. 6.13 Hold the gold pin end of the gold pin wire with the #5 forceps and pierce the exterior surface of the tube at the right upper 270 • lead mark with the needle. 6.14 Pass the needle through the puncture until reaching the inner wall of the tube across the left 270 • upper lead mark and pierce the inner wall of the tube at that location. 6.15 Pass the entire needle towards the outside of the tube until the uninsulated end of the gold pin wire has slipped out of the eye (Figure 14b).

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries.
2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel (Figure 1c).

Torch Setup (5 min)
This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Hold the gold pin end of the gold pin wire at the outside of the tube while performing step 6.15. 6.16 Insert the 10 µL pipette tip through the anterior cut to press the gold pin wire against the inside wall of the tube. 6.17 Slowly retract the gold pin wire by pulling the gold pin end at the right side of the tube until only uninsulated wire lies against the inside of the tube.

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries.
2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel ( Figure 1c).  into a sharp blue flame. Opening the valve too fast or too much might cause a loud popping sound. If this occurs, close the oxygen valve, reignite the pilot flame, and repeat this step. CRITICAL STEP Keep the blue flame approximately 1 cm away from the wire when performing Step 1.4. Passing the blue flame too close to the PI wire will result in a wire kink that will damage the wire and irritate the vagus nerve after implantation. 1.4 PAUSE STEP Performing Steps 1.4 and 1.5 will heat the micro-alligator clip and the PI wire.
Always allow approximately 1 min for cool down before touching the clip and the wire after these steps. Looking under the microscope, point the tip of the blue sharp flame to the 1cm PI wire end and pass it all the way across without touching it (approximately 1 cm away from the wire), heating it to a bright orange. Repeat this several times until the wires have fused together which will be indicated by the formation of bubbles in the PI wire (Figure 3a,c). 1.5 Looking under the microscope, touch the end tip of the clamped 1 cm PI wire with the sharp blue flame to melt 1 mm of the PI wire into a ball. We will refer to the small ball and the fused 9 mm of wire collectively as the "small ball end". CRITICAL STEP Close the oxygen and propane valves at the handle of the jewelry torch. Place the jewelry torch in a secured location until further indication. Release the PI wire from the alligator clip and use a ruler to measure 7.7 cm of PI wire from the melted ball of the small ball end to the spool of PI wire. Then, cut the wire at that location using the surgical scissors. 1.6 Use a ruler to measure 8 mm of PI wire at the insulated end of the 7.7 cm PI wire (opposite from the small ball end). Secure the wire in the micro-alligator clip. 1.7 Completely melt the 8 mm of PI wire into a ball by re-igniting the jewelry torch and touching the 8 mm PI wire end with the sharp blue flame. We will refer to this melted section as the "big ball end". Completion of Steps 1-1.8 in this section will create a 6.9 cm wire with a big ball end, a 6 cm insulated section (middle portion), and a small ball end (9 mm of fused wire with small ball) (Figure 3b).
PAUSE STEP Put UV goggles on. 6.18 Apply a small drop of UV glue to the puncture at the left 270 • upper mark using the eye of a clean needle and cure it for ten seconds under UV light. 6.19 Repeat Steps 6.12-6.18 using the second 270 • gold pin wire at the right and left lower 270 • marks. 6.20 Release the upper two silk strands from the tape and flip the 6 mm tube to move the posterior side facing up. There is no need to reattach the silk strands to the working surface. 6.21 Insert the uninsulated end of one of the 120 • gold pin wires through the eye of the needle. This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Do not loop or knot the wire to the needle at any point. 6.22 Use the needle to pierce the exterior surface of the tube at the upper right 120 • mark towards the anterior cut. 6.23 Pass the needle completely through the puncture leaving the gold pin end of the gold pin wire at the outside of the tube. 6.24 Make a U-turn with the needle and use it to pierce the interior wall of the tube directly across the upper left 120 • mark. Once the needle is completely through the puncture, release the gold pin wire from its eye. This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.

CRITICAL STEP
The gold pin wire should form a D loop at the inside of the tube between the right and left upper 120 • marks. 6.25 Insert the pipette tip through the anterior cut to press the gold pin wires at the inside of the tube (Figure 14c). 6.26 Retract the 120 • gold pin wire by pulling the gold pin end at the right upper 120 • mark until only the uninsulated portion remains on the inside of the tube. Step 1.4. Passing the blue flame too close to the PI wire will result in a wire kink that will damage the wire and irritate the vagus nerve after implantation. 1.4 PAUSE STEP Performing Steps 1.4 and 1.5 will heat the micro-alligator clip and the PI wire.
Always allow approximately 1 min for cool down before touching the clip and the wire after these steps. Looking under the microscope, point the tip of the blue sharp flame to the 1cm PI wire end and pass it all the way across without touching it (approximately 1 cm away from the wire), heating it to a bright orange. Repeat this several times until the wires have fused together which will be indicated by the formation of bubbles in the PI wire (Figure 3a,c). 1.5 Looking under the microscope, touch the end tip of the clamped 1 cm PI wire with the sharp blue flame to melt 1 mm of the PI wire into a ball. We will refer to the small ball and the fused 9 mm of wire collectively as the "small ball end".
CRITICAL STEP Close the oxygen and propane valves at the handle of the jewelry torch.
Place the jewelry torch in a secured location until further indication. Release the PI wire from the alligator clip and use a ruler to measure 7.7 cm of PI wire from the melted ball of the small ball end to the spool of PI wire. Then, cut the wire at that location using the surgical scissors. 1.6 Use a ruler to measure 8 mm of PI wire at the insulated end of the 7.7 cm PI wire (opposite from the small ball end). Secure the wire in the micro-alligator clip. 1.7 Completely melt the 8 mm of PI wire into a ball by re-igniting the jewelry torch and touching the 8 mm PI wire end with the sharp blue flame. We will refer to this melted section as the "big ball end". Completion of Steps 1-1.8 in this section will create a 6.9 cm wire with a big ball end, a 6 cm insulated section (middle portion), and a small ball end (9 mm of fused wire with small ball) (Figure 3b).
PAUSE STEP Put UV goggles on. 6.27 Apply a small drop of UV glue to the puncture at the left upper 120 • mark using the eye of a clean needle and cure it for ten seconds under UV light. Then, remove the pipette tip at the inside of the tube. 6.28 Repeat Steps 6.21-6.27 with the second 120 • gold pin wire at the right and left lower 120 • marks. 6.29 Repeat Steps 6.21-6.28 using the two 60 • gold pin wires at the upper and lower right and left 60 • marks (Figure 14d).

7
Cuff Knotting (10 min). Use the silk-threaded needle to pierce the exterior surface of the tube (and through the anterior cut) 0.5 mm below the upper 270 • mark at the posterior side of the tube. Continue pulling the needle until approximately 3 cm of silk are left on the outside. We will refer to the 3cm silk piece as the short silk end.

7.1
Turn the needle 180 • , pass it through the anterior cut, and pierce the inside surface of the tube approximately 0.5 mm below the upper 60 • electrode. Continue pulling the needle towards the outside of the tube until a "D" loop has formed at the inside of the tube covering the 270 • lead and the 120 • lead.

Preparation of Tools
Drilling Tool (5 min). This section details the process of making a tool to drill holes in the polyurethane tube, through which leads and silk thread will be passed to construct the cuff. Handle the sharp edge with caution. Repeat the third and fourth step in this section to sharpen the drilling tool as needed during the micro-construction procedure (approximately every two uses). 1 Hold the ends of a 22G 1" needle using the alligator clips of two helping hands and cut 1.5 cm of the needle shaft using wire cutters (Figure 1a). Dispose of the sharp needle tip in a sharps container. CRITICAL STEP Use caution when handling either end to prevent injuries.
2 Hold the tip of the needle perpendicular to the surface of a metallic file and file the tip of the needle until completely flat. 3 Hold the flat needle tip against the metallic nail file at a 45° angle and move the needle sideways while rotating it to sharpen the edges (FIGURE 1b). 4 Place the needle under a microscope (lumen facing up) and remove any metallic residues at the lumen by inserting and rotating the tip of the #11 scalpel blade into the flat bevel (Figure 1c). This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP The long piece of silk going towards the outside of the tube (with the needle at the end) will be referred to as the long silk end. 7.2 Make a ligature knot at the outside of the tube using the short and long silk ends. 7.3 Use the scissors to trim both ends of the ligature knot to approximately 3 mm. Step 1.4. Passing the blue flame too close to the PI wire will result in a wire kink that will damage the wire and irritate the vagus nerve after implantation.
PAUSE STEP Wear UV goggles for the following steps. 7.4 Apply a small drop of UV glue to the ligature knot with the eye of a clean needle and cure it by shining UV light over the glue for ten seconds. The UV glue drop should be only big enough to cover the knot.

7.5
Repeat Steps 7-7.4 0.5 mm below the lower 270 • mark and 0.5 mm below the lower 60 • electrode at the inside of the cuff. 7. 6 Place the pipette tip inside the tube to press the uninsulated segment of the PI wires against the inner surface of the tube. 7.7 Apply a small drop of UV glue to the right upper and lower 270 • , 120 • , and 60 • punctures with exiting PI wire and cure them under UV light for 10 s. This is now a completed Variable Multi-contact Circumferential Cuff. 7.8 VNS Cuff Impedance Testing (15-20 min) 1 Fill a small container with saline. 2 Connect the test probe pins male plugs to the LCR meter. 3 Hold the gold pins of the cuff in the test probe pins of the LCR meter. 4 Submerge the tube of the cuff completely in the saline. This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Do not submerge the probes with the pins. This section details the process of setting up a jewelry torch, which is necessary to construct the wires which are for the stimulation contacts in the cuff.
CRITICAL STEP Use caution and follow manufacturer instructions when setting up the torch. 1 Connect the oxygen and propane preset regulators to the fuel hoses of the jewelry torch by capping their adapters.
CRITICAL STEP Visually ensure that saline fills the inside of the cuff. 5 Turn on the LCR meter and set the frequency to 1 kHz, then observe the impedance value displayed on the screen. Functional VNS cuffs should have impedance of less than 2 kΩ.  Cuff Knotting (10 min). Use the silk-threaded needle to pierce the exterior surface of the tube (and through the anterior cut) 0.5 mm below the upper 270° mark at the posterior side of the tube. Continue pulling the needle until approximately 3 cm of silk are left on the outside. We will refer to the 3cm silk piece as the short silk end. 7.1 Turn the needle 180°, pass it through the anterior cut, and pierce the inside surface of the tube approximately 0.5 mm below the upper 60° electrode. Continue pulling the needle towards the outside of the tube until a "D" loop has formed at the inside of the tube covering the 270° lead and the 120° lead. CRITICAL STEP The long piece of silk going towards the outside of the tube (with the needle at the end) will be referred to as the long silk end. 7.2 Make a ligature knot at the outside of the tube using the short and long silk ends. 7.3 Use the scissors to trim both ends of the ligature knot to approximately 3 mm. PAUSE STEP Wear UV goggles for the following steps. 7.4 Apply a small drop of UV glue to the ligature knot with the eye of a clean needle and cure it by shining UV light over the glue for ten seconds. The UV glue drop should be only big enough to cover the knot. 7.5 Repeat Steps 7-7.4 0.5 mm below the lower 270° mark and 0.5 mm below the lower 60° electrode If the cuff contains more than two gold pins, switch the gold pins on the probe pins of the LCR meter and record their impedance.

Expected Results
We used two methods to confirm the efficacy of nerve stimulation using cuffs constructed using the above described protocols. First, to evaluate activation of the vagus nerve, we measured rapid stimulation-dependent depression of blood oxygen saturation, an effect ascribed to the Hering-Breuer reflex [26]. Stimulation of vagal A-fibers, which include the pulmonary stretch receptors, generates a feedback suppression of inhalation and causes blood oxygen saturation to transiently fall (Figure 15a). Therefore, measurement of oxygen saturation provides a simple means to assess vagal A-fiber recruitment. As expected, 10 s trains of stimulation (0.8 mA, 100 µs biphasic pulses, 30 Hz) delivered using the standard nerve cuff model generate robust, reliable drops blood oxygen saturation, signaling activation of the vagus nerve ( Figure 15a). saturation, signaling activation of the vagus nerve (Figure 15a).
We next tested activation of the sciatic nerve using the longitudinal and multichannel cuffs. Stimulation of the A-fibers in the sciatic nerve triggers contraction of the gastrocnemius muscle, thus assessment of the degree of force generated by the gastrocnemius provides a simple means to evaluate A-fiber recruitment. As expected, stimulation delivered across a range of stimulation intensities (0.5 s train, 0-0.5 mA, 100 µs biphasic pulses, 30 Hz) using either the longitudinal and multichannel circumferential cuffs both produced effective recruitment of the nerve at low stimulation intensities (Figure 15b). Together, these data indicate that the cuff construction methods above provide reliable vagal and sciatic nerve stimulation. Conflicts of Interest: M.P.K. is a consultant for, and has a financial interest in, MicroTransponder, Inc., which is developing therapies using VNS. C.T.E. is married to an employee of MicroTransponder, Inc. All other authors report no conflicts of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the Defense Advanced Research Projects Agency (DARPA) Biological Technologies Office (BTO).