Fabrication of the Composite Regenerative Peripheral Nerve Interface (C-RPNI) in the Adult Rat

The following manuscript describes a novel method for developing a biologic, closed loop neural feedback system termed the composite regenerative peripheral nerve interface (C-RPNI). This construct has the ability to integrate with peripheral nerves to amplify efferent motor signals while simultaneously providing afferent sensory feedback.

Our protocol is significant in that it allows simultaneous motor nerve signal amplification alongside the provision of afferent sensory nerve stimulation through the utilization of a biologic peripheral nerve interface. This technique can provide a life-like prosthetic device for those with amputations as the C-RPNI allows simultaneous motor control and sensory feedback within the same peripheral nerve interface. Many of those living with amputations abandon advanced neuroprothestic devices because the devices lack intuitive control and meaningful sensory feedback.

This technique provides both and can prevent prosthetic device abandonment. This technique could be applied to individuals with either weak or absent limbs or sensory deficits providing further insight into feedback systems that provide precise and intuitive extremity control. Performing surgery on peripheral nerves is difficult as there is little margin for error.

Using high-quality instruments and practicing often are the best ways to ensure success. Surgical techniques are incredibly difficult to convey and to learn from a literary format. Observing each and every step involved in the construct fabrication is key to mastering the method.

To prepare a skin graft, after confirming a lack of response to toe pinch, apply ointment to the eyes of the anesthetized rat. Using clippers, shave the entire lower hindlimb, ankle region and sides of the paw. Disinfect the selected hindlimb in the plantar surface of the paw with a sequential alcohol-iodopovidone solution and alcohol cleanse.

Using a handheld micro motor high-speed drill with a removable round fine grit polishing stone, burr the plantar surface of the paw to remove the epidermis at a speed of 4, 000 revolutions per minute applying drops of saline so as not to burn the skin. The underlying dermis will have a shiny appearance with pinpoint bleeding. Apply a tourniquet to the lower extremity to slow the blood flow and use a number 15 scalpel to sharply remove the plantar skin.

Place the skin in saline moistened gauze to prevent desiccation and apply a gauze wrap to the bleeding foot to slow hemorrhage. Place the skin under a dissecting microscope and use microscissors to remove any tendinous and connective tissue from the deep layer of the skin graft. The thinned dermal graft should be slightly opaque, contain only dermis and measure approximately 0.5 by one centimeter in size.

Then place the tissue into a new piece of saline moistened gauze until construct fabrication. To prepare a muscle graft, use a number 15 scalpel to make a longitudinal incision along the anterior aspect of the lower hindlimb from just above the ankle to just below the knee. Dissect through the subcutaneous tissue to expose the underlying musculature.

At the distal aspect of the incision, expose the tendinous insertions of the lower limb musculature. The tibialis anterior is typically the largest and most anterior of the muscles. Just underneath and posterior to this muscle lies the extensor digitorum longus.

To isolate the distal extensor digitorum longus tendon from the other tendons in the area, insert both tines of a forceps underneath the tendon and open the forceps to exsert upward pressure to cause tendon excursion. Manipulation of the tendon should cause all of the toes to extend simultaneous. Use sharp iris scissors to perform a distal tenotomy and use tenomoties to bluntly separate the muscle from the surrounding tissue working proximally to find the tendinous origin.

Once the proximal tendon can be visualized, use the iris scissors to perform a second tenotomy and place the muscle graft in a saline moistened gauze. For isolation and preparation of the common peroneal nerve, shave one thigh and disinfect the exposed skin with a sequential alcohol-Betadine-alcohol cleanse. Place the anesthetized rat onto a heating pad under a surgical microscope and mark the incision from just distal to the sciatic notch to the inferior portion of the knee inferior to and angled away from the femur.

Then use a number 15 scalpel to make an incision along the mark through the underlying biceps femoris fascia. Carefully dissect through the biceps femoris muscle to the space underlying the biceps femoris. Following the identification of the common peroneal nerve, use micro fine-tipped forceps and microscissors to carefully isolate the common peroneal nerve from the other sciatic branches.

Remove any lingering connective tissue distally and at the point at which the nerve crosses the surface of the knee, sharply transect the nerve with a pair of microscissors. Then carefully free any remaining connective tissue from the common peroneal nerve and work proximally to free the nerve to a length of approximately two centimeters. Nerves are delicate and minor missteps can cause lasting functional deficits.

Frequent practice, precise surgical instruments, and ideal hand positioning and support are key to mastering this technique. To fabricate the C-RPNI construct, place the muscle graft under the dissecting microscope and remove all of the central tendinous tissue as well as a small central segment of epimysium leaving the tendinous ends intact. Using an 8-0 nylon suture and two interrupted stitches, secure the epineurium of the transected end of the common peroneal nerve to the area of the muscle graft devoid of epimysium on either side of the nerve.

Secure the muscle graft to the femur periosteum with a single 6-0 nylon interrupted stitch both proximally and distally with the nerve muscle junction facing away from the femur. Place an 8-0 nylon stitch at the inferior central margin of the muscle graft epimysium securing it to the common peroneal nerve epineurium so as to create laxity in the nerve within the muscle graft. Position the skin graft on the muscle graft so that it completely covers the nerve and the majority of the muscle with the deep margin of the dermis resting on the muscle.

Trim any dermis that extends beyond the border of the muscle and use 8-0 nylon interrupted sutures to circumferentially secure the skin graft to the muscle graft. Close the biceps femoris fascia over the construct in a running fashion with 5-0 chromic suture and close the overlying skin with a 4-0 chromic suture in running fashion. Then swap the surgical area with an alcohol pad and apply antibiotic ointment before allowing the rat to recover with food and water sources separate from cage mates with monitoring until full recumbency.

If successful, surgical exposure of the constructs after three months will reveal re-vascularized muscle and skin and gentle squeezing of the common peroneal nerve with forceps proximal to the construct will result in visible muscle contraction. Histological analysis should demonstrate viable skin, nerve, and muscle. Immunostaining will also reveal motor and sensory nerve re-innervation to their neuromuscular junctions and sensory end organs respectively.

Electrophysiologic testing can be performed on these constructs in vivo as demonstrated, for example at three and nine months following C-RPNI fabrication. Here, single and summation compound muscle action potentials and compound sensory nerve action potentials signals obtained during electrophysiologic testing in a graphical format are shown. Suboptimal results at the muscle graft are indicated by attenuated signals that lack the characteristic compound muscle action potential waveform.

Suboptimal results at the level of the dermal component usually involve dampening of the waveform with significant background noise. Good quality instruments are vital. Scissors should be sharp and forceps should be fine tipped to facilitate an extremely precise handling of the dermal grafts and nerve without damage.

Following fabrication, this method can be utilized in electrophysiological investigations to further characterize signaling capabilities providing more insight into efferent and afferent feedback loops. Although the C-RPNI allows for sensory feedback, it's unclear whether a closed-loop feedback system for proprioception can be established. We're currently addressing this question in the lab.