Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

Peripheral nerve stimulation is increasingly under investigation as a treatment for a variety of diseases including inflammatory diseases and neural disorders. Construction of chronically implantable peripheral nerve cuff electrodes for small laboratory animals can be time consuming and expensive. This protocol describes a simple, low cost approach for constructing chronically implantable peripheral nerve stimulating electrodes for use in rats.

The method requires very few and easily accessible materials and supplies and a reduced number of hazardous or technically demanded steps compared to similar techniques which reduces both the cost of the device as well as the time required to train personnel and device assembly. This technique was designed to enable preclinical studies that test the effectiveness of chronic Vegas nerve stimulation during stroke recovery or other types of Metro dysfunction. Begin by preparing the cuff tubing.

Use a razor blade to cut a 2.5 millimeter piece of polymer tubing. Then insert a paperclip or force tips through the tubing and make a lengthwise slip through the wall of the tubing on one side of the cuff. Remove the forceps from the tubing and insert a large sewing needle through the midline of the cuff.

Place the needle into the foam board to pin the cuff in place during the remaining assembly steps. To place a suture for securing cuff closure during implantation insert a small sewing needle through the wall of the cuff on the midline approximately point five millimeters from the top slit on one side. Then insert two centimeters of a six zero suture through the eye of the needle and pull the needle through the wall of the tubing to thread the suture into the cuff.

Puncture a second hole through the tubing wall approximately point five millimeters below the first hole along the midline of the cuff. Insert the suture through the eye of the needle and pull the needle through the tubing wall. Apply a small amount of UV cure adhesive to the short end of the suture extending from the lower hole and pull the longer suture end until the lower tail is nearly flush with the exterior wall of the tubing.

Then cure the adhesive with a UV wand. Repeat this process to place another suture on the opposite side of the cuff. Next place the Platinum Iridium wire leads.

Use the small sewing needle to make four holes in the cuff wall. Then insert the sewing needle again, this time working from exterior to interior through the first lead hole. Insert approximately point five centimeters of a 7.5 centimeter Platinum Iridium wire through the eye of the needle and pull the needle through the tubing to thread the wire lead through the cuff wall.

Adjust the wire so that approximately 4.5 centimeters extends on the exterior side of the cuff. Insert the needle through the first lead hole again then through the second lead hole. Insert point five centimeters of the shorter end of the wire through the eye of the needle and pull the needle through the tubing to thread the wire lead through the cuff walls.

Repeat the process to thread the wire through the third and fourth lead holes. Using a butane lighter, carefully remove the insulation from five to six millimeters of the wires extending from the second and fourth lead holes Then gently pull on the end of the wire extending from the first hole until the uninsulated portion of the wire is flush with the hole. Repeat this with the other lead to align the uninsulated end of the wire threaded through the third and fourth lead holes.

Apply a small amount of UV cure adhesive to the wire loops on the exterior side of the cuff at the first and third lead holes. Then use the UV wand to cure of the adhesive and secure the leads in place. Use a small pipette tip to push the uninsulated wire leads against the interior wall of the cuff.

Pull the one millimeter tails of the wire flat against the exterior surface of the cuff, taking care not to short them together. Then apply a small amount of UV adhesive to just cover the two tails and cure it. Remove the large needle with the cuff assembly from the foam board.

Insert a three centimeter length of a six zero suture through the eye of the needle and pull the needle through the tubing to thread the suture through the bottom of the cuff at the midpoint. Insert the small sewing needle through the same midline hole working from interior to exterior to avoid deformation, the tubing and the wire leads. Then insert the exterior tail of the suture through the eye of the needle and pull the needle through the cuff wall to create a loop of suture around the edge of the cuff.

Create the second loop around the opposite end of the cuff by tying the ends of the suture and a half knot on the exterior side of the cuff. While holding the knot flat against the tubing, apply a small amount of UV adhesive to the half knot and cure it. Carefully cut the ends of the suture thread as close to the knot as possible.

Then, use a butane lighter to remove the insulation from approximately three millimeters at the end of each of the Platinum Iridium wire leads. Solder the cup side of a gold pin to the uninsulated end of each lead. Before proceeding with implantation test the impedance of the assembled device to verify that the assembled cuff has an impedance of less than two kilo ohm at one kilohertz.

After assembling the head cap and implanting the cuff electrodes according to manuscript directions, stimulate the vagus nerve while the rat is awake. Connect the rat to a stimulus generator via the implanted head cap and adjust the stimulation settings. For VNS induced reorganization of the motor cortical map, deliver a single train of 15 biphasic pulses, each with a width of 100 microseconds, and an amplitude of 800 micro ampere at a frequency of 30 hertz.

A stimulation train is delivered immediately after the detection of each successful lever press throughout 10, 30 minute training sessions. Use an oscilloscope to monitor successful delivery of current stimulation. This protocol, was used to chronically implant vagus nerve cuff electrodes and head caps in rats.

During implantation, functional validation of all cuffs was performed by testing for a VNS induced drop in blood oxygen saturation. To evoke this response, a 10 second train of 30 hertz pulses, each with a width of 100 microseconds, and an amplitude of 800 micro ampere was delivered across the cuff leads. For all cuffs, we observed a VNS induced cessation of breathing for the duration of the 10 seconds stimulation, which was accompanied by a drop in blood oxygen saturation of at least 5%confirming cuff function and proper implantation.

To test the long term functionality of the devices, rats were anesthetized six weeks after implantation, and VNS was again applied. For seven out of nine devices, the magnitude of stimulation evoked change in blood oxygen saturation, did not differ from that observed at initial implantation, suggesting continued excellent performance. The remaining two devices, increasing the stimulation amplitude to 1.6 milliampere was sufficient to evoke a reliable reduction in blood oxygen saturation of at least 5%suggesting that these devices continued to function.

But the changes in impedance, nerve damage, or cuff orientation may have resulted in reduced performance. To further test the long term functionality of the chronically implanted devices, a second group of rats was trained on a simplified version of a skilled reaching lever press task. The rats were required to reach two centimeters outside the training booth to fully depress the lever and then to release it within two seconds.

Consistent with prior studies demonstrating that vagus nerve stimulation drives expansion of task relevant motor map representations, VNS treated rats exhibited significantly larger proximal forelimb representations than Sham treated rats. This protocol can be easily adapted to accommodate chronic stimulation experiments in other laboratory species or at other peripheral nerve sites, including sciatic or sacral nerves. For example.