The authors wish to thank Jana Moon for expert technical assistance. Studies presented in this paper were funded through an R21 (R21NS104584) grant to SK.

All animal experiments are performed under the approval of the University of Michigan&#39;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 ...

<ol>
	<li>Biddiss, E. A., Chau, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upper+limb+prosthesis+use+and+abandonment:+A+survey+of+the+last+25+years.">Upper limb prosthesis use and abandonment: A survey of the last 25 years.</a> Prosthetics and Orthotics International. 31 (3), 236-257 (2007).</li><li>Kung, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+peripheralnerve+interface+viability+and+signal+transduction+with+an+implanted+electrode.">Regenerative peripheralnerve interface viability and signal transduction with an implanted electrode.</a> Plastic and Reconstructive Surgery. 133 (6), 1380-1394 (2014).</li><li>Larson, J. 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The effects of prolonged constriction on the distal nerve segment.</a> Muscle Nerve. 12 (11), 915-928 (1989).</li><li>Micera, S., Navarro, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+interfaces+with+the+peripheral+nervous+system.">Bidirectional interfaces with the peripheral nervous system.</a> International Review of Neurobiology. 86, 23-38 (2009).</li><li>Urbanchek, M. 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The authors have no disclosures.

The C-RPNI is a novel construct that provides simultaneous amplification of a target nerve&#39;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 ...

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 extremity4,5. 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 &#34;advanced prosthetics&#34; difficult to learn and therefore not suitable for everyday use1,6. 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 cues7,8,9. For these reasons, advanced prostheses are associated with a significant degree of mental fatigue and are often described as burdensome and unsatisfactory5,10,11. To address this, some research laboratories have developed prosthetics capable of providing a limited degree of sensory feedback via direct neural interaction12,13,14,15, but feedback is often limited to small, scattered areas on the hands and fingers12,13, 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&#39;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 plates14,15,18, extra-neural cuff electrodes13,19,20,21,22,23, tissue penetrating electrodes24,25,31,32, and intrafascicular electrodes26,27,28. 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 response16,17,18,19. More recently, it has been postulated that a driver behind eventual implanted electrode failure is the significant difference in Young&#39;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&#39;s moduli causes inflammation and eventual permanent scarring30,31,32. 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 freedom2,34. 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.

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

fabrication of the composite regenerative peripheral nerve interface (c-rpni) in the adult rat

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&#39;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.

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 reinnervate2,3,17,36. Following the three-month recovery period, construct testing can be pursued to examine viability. Surgical exposure of t...

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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.

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

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is ...

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.

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Research

JoVE Journal

Bioengineering

8.0K Views.  University of Michigan, Ann Arbor. 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 b...