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

Summary

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.

Abstract

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is often accomplished through integration with the peripheral nervous system. Unfortunately, methods currently employed are often associated with significant tissue damage which prevents prolonged use. Additionally, these devices often lack any meaningful degree of sensory feedback as their complex construction dampens any vibrations or other sensations a user may have previously depended on when using more simple prosthetics. The composite regenerative peripheral nerve interface (C-RPNI) was developed as a stable, biologic construct with the ability to amplify efferent motor nerve signals while providing simultaneous afferent sensory feedback. The C-RPNI consists of a segment of free dermal and muscle graft secured around a target mixed sensorimotor nerve, with preferential motor nerve reinnervation of the muscle graft and sensory nerve reinnervation of the dermal graft. In rats, this construct has demonstrated the generation of compound muscle action potentials (CMAPs), amplifying the target nerve's signal from the micro- to milli-volt level, with signal to noise ratios averaging approximately 30-50. Stimulation of the dermal component of the construct generates compound sensory nerve action potentials (CSNAPs) at the proximal nerve. As such, this construct has promising future utility towards the realization of the ideal, intuitive prosthetic.

Introduction

Extremity amputations affect nearly 1 in 190 Americans¹, and their prevalence is projected to increase from 1.6 million today to over 3.6 million by 2050². Despite documented use for over a millennium, the ideal prosthetic has yet to be realized³. Currently, there exist complex prosthetics capable of multiple joint manipulations with the potential to reproduce many motor functions of the native extremity^4,5. However, these devices are not considered intuitive as the desired prosthetic motion is typically functionally separate from the input control signal. Users typically consider these "advanced prosthetics" difficult to learn and therefore not suitable for everyday use^1,6. Additionally, complex prosthetics currently on the market do not provide any appreciable degree of subtle sensory feedback for adequate control. The sense of touch and proprioception are vital to carrying out daily tasks, and without these, simple acts such as picking up a cup of coffee become burdensome as it relies entirely on visual cues^7,8,9. For these reasons, advanced prostheses are associated with a significant degree of mental fatigue and are often described as burdensome and unsatisfactory^5,10,11. To address this, some research laboratories have developed prosthetics capable of providing a limited degree of sensory feedback via direct neural interaction^12,13,14,15, but feedback is often limited to small, scattered areas on the hands and fingers^12,13, and sensations were noted to be painful and unnatural at times¹⁵. Many of these studies unfortunately lack any appreciable long-term follow-up and nerve histology to delineate local tissue effects, while noting interface failure on the scale of weeks to months¹⁶.

For this population, the ideal prosthetic device would provide high fidelity motor control alongside meaningful somatosensory feedback from the individual's environment throughout their lifetime. Critical to the design of said ideal prosthetic is the development of a stable, reliable interface that would allow for simultaneous transmission of afferent somatosensory information with efferent motor signals. The most promising of current human-machine interfaces are those that interact with the peripheral nervous system directly, and recent developments in the field of neuro-integrated prosthetics have worked towards bridging the gap between bioelectric and mechanical signals¹⁷. Current interfaces utilized include: flexible nerve plates^14,15,18, extra-neural cuff electrodes^{13,19,20,21,22,23}, tissue penetrating electrodes^24,25,31,32, and intrafascicular electrodes^26,27,28. However, each of these methods has demonstrated limitations with regards to nerve specificity, tissue injury, axonal degeneration, myelin depletion, and/or scar tissue formation associated with chronic indwelling foreign body response^16,17,18,19. More recently, it has been postulated that a driver behind eventual implanted electrode failure is the significant difference in Young's moduli between electronic material and native neural tissue. Brain tissue is subject to significant micromotion on a daily basis, and it has been theorized that the shear stress induced by differences in Young's moduli causes inflammation and eventual permanent scarring^30,31,32. This effect is often compounded in the extremities, where peripheral nerves are subject to both physiologic micromotion and intentional extremity macromotion. Due to this constant motion, it is reasonable to conclude that utilization of a completely abiotic peripheral nerve interface is not ideal, and an interface with a biologic component would be more suitable.

To address this need for a biologic component, our laboratory developed a biotic nerve interface termed the Regenerative Peripheral Nerve Interface (RPNI) to integrate transected peripheral nerves in a residual limb with a prosthetic device. RPNI fabrication involves surgically implanting a peripheral nerve into an autologous free muscle graft, which subsequently revascularizes and reinnervates. Our lab has developed this biologic nerve interface over the past decade, with success in amplifying and transmitting motor signals when combined with implanted electrodes in both animal and human trials, allowing for suitable prosthetic control with multiple degrees of freedom^2,34. In addition, we have separately demonstrated sensory feedback through the use of peripheral nerves embedded in dermal grafts, termed the Dermal Sensory Interface (DSI)^3,35. In more distal amputations, using these constructs simultaneously is feasible as motor and sensory fascicles within the target peripheral nerve can be surgically separated. However, for more proximal level amputations, this is not feasible due to intermingling of motor and sensory fibers. The Composite Regenerative Peripheral Nerve Interface (C-RPNI) was developed for more proximal amputations, and it involves implanting a mixed sensorimotor nerve into a construct consisting of free muscle graft secured to a segment of dermal graft (Figure 1). Peripheral nerves demonstrate preferential targeted reinnervation, thus sensory fibers will re-innervate the dermal graft and motor fibers, the muscle graft. This construct thus has the ability to simultaneously amplify motor signals while providing somatosensory feedback³⁶ (Figure 2), allowing for the realization of the ideal, intuitive, complex prosthetic.

Protocol

All animal experiments are performed under the approval of the University of Michigan's Committee on the Use and Care of Animals.

NOTE: Donor rats are allowed free access to food and water prior to skin and muscle donation procedures. Euthanasia is performed under deep anesthesia followed by intra-cardiac potassium chloride injection with a secondary method of bilateral pneumothorax. Any strain of rat can theoretically be utilized with this experiment; however, our laboratory has achieved consistent results in both male and female Fischer F344 rats (~200-250 g) at two to four months of age. Donor rats must be isogenic to the experimental rats.

1. Preparation of the dermal graft

1. Anesthetize donor rat in an induction chamber utilizing a solution of 5% isoflurane in oxygen at 0.8-1 L/min. Once the rat has been anesthetized, remove from induction chamber and place on a rebreathing nose cone, lowering the isoflurane to 2-2.5% for maintenance of anesthesia.
2. Administer a solution of 0.02-0.03 mL Carprofen (50 mg/mL) in 0.2 mL of sterile saline subcutaneously between the shoulder blades for analgesia.
3. Apply artificial tears ointment to both eyes to prevent corneal ulcers.
4. Using clippers, shave the entire lower hindlimb(s), ankle region, and sides of paw(s).
5. Cleanse chosen hindlimb and plantar surface of paw with alcohol, followed by iodopovidone solution, ending with a final cleanse with alcohol to remove residual iodopovidone.
6. Using a hand-held micro motor high speed drill with a removable round fine grit polishing stone (4000 rpm), burr the plantar surface of the paw to remove the epidermis. While burring, apply drips of saline as to not burn the skin. The underlying dermis will have a shiny appearance with pinpoint bleeding.
7. Apply a tourniquet to the lower extremity to slow blood flow.
8. Remove the plantar skin sharply with a #15 scalpel and place in saline-moistened gauze to prevent desiccation. Some tendinous and connective tissue will inherently be removed with the skin in this step and will be removed later.
9. Apply gauze wrap to the bleeding foot to slow hemorrhage. Repeat steps 1.5-1.9 if doing two constructs.
10. Under a microscope (20x magnification), remove the tendinous and connective tissue from the deep layer of the skin graft using micro-scissors. Take care not to make holes in the graft. The thinned dermal graft should be slightly opaque containing only dermis, measuring approximately 0.5 cm x 1.0cm in size.
11. Place in saline-moistened gauze until ready for C-RPNI construct fabrication. Grafts should be utilized within 2 hours of harvest.

2. Preparation of the muscle graft

1. Make a longitudinal incision along the anterior aspect of the lower hindlimb from just above the ankle to just below the knee with a #15 scalpel. Dissect through subcutaneous tissue to expose the underlying musculature.
2. At the distal aspect of the incision, expose the tendinous insertions of the lower limb musculature. Tibialis anterior (TA) is typically the largest and most anterior of the muscles, and just underneath and posterior to this muscle lies the extensor digitorum longus (EDL). Isolate the distal EDL tendon from the other tendons in the area, taking care not to incise its insertion at this point.
3. Ensure isolation of the correct tendon by inserting both tines of a forceps underneath the tendon and exerting upward pressure by opening the forceps to cause tendon excursion. Manipulation of this tendon should cause all of the toes to extend simultaneously.
4. Perform a distal tenotomy with sharp iris scissors and separate the muscle from the surrounding tissues bluntly with tenotomies (or other blunt-tipped scissor) working proximally to find the tendinous origin.
5. Once the proximal tendon is visualized, again perform a tenotomy utilizing sharp iris scissors. Place the muscle graft in a saline-moistened gauze to prevent desiccation.
6. Once all desired grafts have been removed from a donor rat, euthanize primarily by intra-cardiac KCl injection (1-2 mEq K+/kg) followed by secondary euthanasia with bilateral puncture pneumothorax with a #15 blade.

3. Common peroneal nerve isolation and preparation

1. Anesthetize and provide analgesia to the experimental rat according to protocol outlined in steps 1.1-1.3.
2. Shave the desired thigh and cleanse with alcohol, betadine, ending with alcohol to remove traces of betadine.
3. Move animal from surgical prep table to surgical microscope table and place on heating pad with temperature probe for body temperature maintenance. Maintain isoflurane at 2-2.5% and oxygen at 0.8-1 L/min.
4. Mark the incision, extending from just distal to sciatic notch to the inferior portion of the knee. This marking should be inferior to, and angled away from, the femur. Make the incision with a #15 blade incising through the underlying biceps femoris fascia.
5. Carefully dissect through the biceps femoris muscle with either a hemostat or blunt-tipped micro-scissors to the space underlying biceps femoris.
   NOTE: The sciatic nerve travels approximately in the same direction as the initial incision that was made. There are three branches, typically with sural nerve posterior and common peroneal and tibial nerve traveling superficial and deep to the knee, respectively.
6. Following identification of the common peroneal (CP) nerve, using a pair of micro-, fine-tipped forceps and micro-scissors, carefully isolate the CP nerve from the other sciatic branches and remove any lingering connective tissue distally.
7. At the point where the nerve crosses the surface of the knee, sharply transect the nerve with a pair of micro-scissors.
   NOTE: Using sharp scissors is extremely important in this step as causing significant trauma to the nerve could increase the risk of neuroma formation.
8. Carefully free any remaining connective tissue from the CP nerve and work proximally to free the nerve to a length of approximately 2 cm.

4. C-RPNI construct fabrication

1. Remove the muscle graft from saline-moistened gauze and remove all central tendinous tissue as well as a small central segment of epimysium. Leave the tendinous ends intact.
2. Using an 8-0 nylon suture, secure the epineurium of the transected end of the CP nerve to the area of the muscle graft devoid of epimysium with two interrupted stitches on either side of the nerve.
3. 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.
   NOTE: Secure the muscle so that it is at normal relaxed length. Try not to stretch the muscle significantly or leave too much laxity when securing.
4. Place an 8-0 nylon stitch at the inferior, central margin of the muscle graft epimysium, securing it to the CP nerve epineurium in a way as to create laxity in the nerve within the muscle graft and help to relieve any future tension it may be exposed to with later ambulation.
5. Remove the skin graft from the saline-moistened gauze and arrange it on the muscle graft in such a way to completely cover the nerve and the majority of the muscle. Ensure that the deep margin of the dermis is resting on the muscle. Trim any dermis that extends beyond the border of the muscle.
6. Secure the skin graft to the muscle graft circumferentially using 8-0 nylon interrupted sutures. Typically, 4-8 total sutures are used depending on the size of the construct.
7. Close the biceps femoris fascia over the construct in a running fashion with 5-0 chromic suture.
8. Close the overlying skin with 4-0 chromic suture in running fashion.
9. Swab the surgical area with an alcohol pad and apply antibiotic ointment.
10. Cease inhalational anesthetic and allow rat to recover with food and water sources separate from cage mates.

Results

Construct fabrication is considered unsuccessful if rats develop an infection or do not survive surgical anesthesia. Previous research has indicated these constructs require approximately three months to revascularize and reinnervate^2,3,17,36. Following the three-month recovery period, construct testing can be pursued to examine viability. Surgical exposure of t...

Discussion

The C-RPNI is a novel construct that provides simultaneous amplification of a target nerve's motor efferent signals with provision of afferent sensory feedback. In particular, the C-RPNI has unique utility for those living with proximal amputations as their motor and sensory fascicles cannot easily be mechanically separated during surgery. Instead, the C-RPNI utilizes the inherent preferential reinnervation properties of the nerve itself to encourage sensory fiber reinnervation to dermal sensory end organs and motor ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
#15 Scalpel	Aspen Surgical, Inc	Ref 371115	Rib-Back Carbon Steel Surgical Blades (#15)
4-0 Chromic Suture	Ethicon	SKU# 1654G	P-3 Reverse Cutting Needle
5-0 Chromic Suture	Ethicon	SKU# 687G	P-3 Reverse Cutting Needle
6-0 Ethilon Suture	Ethicon	SKU# 697G	P-1 Reverse Cutting Needle (Nylon suture)
8-0 Monofilament Suture	AROSurgical	T06A08N14-13	Black polyamide monofilament suture on a threaded tapered needle
Experimental Rats	Envigo	F344-NH-sd	Rats are Fischer F344 Strain
Fluriso (Isofluorane)	VetOne	13985-528-40	Inhalational Anesthetic
Micro Motor High Speed Drill with Stone	Master Mechanic	Model 151369	Handheld rotary tool; kit comes with multiple fine grit stones
Oxygen	Cryogenic Gases	UN1072	Standard medical grade oxygen canisters
Potassium Chloride	APP Pharmaceuticals	63323-965-20	Injectable form, 2 mEq/mL
Povidone Iodine USP	MediChoice	65517-0009-1	10% Topical Solution, can use one bottle for multiple surgical preps
Puralube Vet Opthalmic Ointment	Dechra	17033-211-38	Corneal protective ointment for use during procedure
Rimadyl (Caprofen)	Zoetis, Inc.	NADA# 141-199	Injectable form, 50 mg/mL
Stereo Microscope	Leica	Model M60	User can adjust magnification to their preference
Surgical Instruments	Fine Science Tools	Various	User can choose instruments according to personal preference or from what is currently available in their lab
Triple Antibiotic Ointment	MediChoice	39892-0830-2	Ointment comes in sterile, disposable packets
VaporStick 3	Surgivet	V7015	Anesthesia tower with space for isofluorane and oxygen canister
Webcol Alcohol Prep	Coviden	Ref 6818	Alcohol prep wipes; use a new wipe for each prep