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 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
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Apply artificial tears ointment to both eyes to prevent corneal ulcers.</li>
	<li>Using clippers, shave the entire lower hindlimb(s), ankle region, and sides of paw(s).</li>
	<li>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.</li>
	<li>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.</li>
	<li>Apply a tourniquet to the lower extremity to slow blood flow.</li>
	<li>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.</li>
	<li>Apply gauze wrap to the bleeding foot to slow hemorrhage. Repeat steps 1.5-1.9 if doing two constructs.</li>
	<li>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.</li>
	<li>Place in saline-moistened gauze until ready for C-RPNI construct fabrication. Grafts should be utilized within 2 hours of harvest.</li>
</ol>
2. Preparation of the muscle graft
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Common peroneal nerve isolation and preparation
<ol>
	<li>Anesthetize and provide analgesia to the experimental rat according to protocol outlined in steps 1.1-1.3.</li>
	<li>Shave the desired thigh and cleanse with alcohol, betadine, ending with alcohol to remove traces of betadine.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Carefully free any remaining connective tissue from the CP nerve and work proximally to free the nerve to a length of approximately 2 cm.</li>
</ol>
4. C-RPNI construct fabrication
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Close the biceps femoris fascia over the construct in a running fashion with 5-0 chromic suture.</li>
	<li>Close the overlying skin with 4-0 chromic suture in running fashion.</li>
	<li>Swab the surgical area with an alcohol pad and apply antibiotic ointment.</li>
	<li>Cease inhalational anesthetic and allow rat to recover with food and water sources separate from cage mates.</li>
</ol>

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<li>Carr, M. M., Best, T. J., Mackinnon, S. E., Evans, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strain+differences+in+autotomy+in+rats+undergoing+sciatic+nerve+transection+or+repair%5BTitle%5D)+AND+%22Annals+of+Plastic+Surgery%22%5BJournal%5D)">Strain differences in autotomy in rats undergoing sciatic nerve transection or repair.</a> Annals of Plastic Surgery. 28 (6), 538-544 (1992).</li>
</ol>

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><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...

Watch this Scientific Journal Video about Fabrication of the Composite Regenerative Peripheral Nerve Interface C-RPNI in the Adult Rat at JoVE.com

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 ...

fabrication-composite-regenerative-peripheral-nerve-interface-c-rpni

Research

JoVE Journal

Bioengineering

8.2K Views.  University of Michigan, Ann Arbor. Ons protocol is significant in dat het gelijktijdige motorische zenuwsignaal versterking naast de levering van afferent sensorische zenuwstimulatie door het gebruik van een biologische perifere zenuwinterface mogelijk maakt. Deze techniek kan een levenselig protheseapparaat bieden voor mensen met amputaties, omdat de C-RPNI gelijktijdige motorische controle en sensorische feedback mogelijk maakt binnen dezelfde perifere zenuwinterface. Veel van degenen die leven met amputaties verlaten geavan...

Video: Fabrication of the Composite Regenerative Peripheral Nerve Interface C-RPNI in the Adult Rat

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Combining Peripheral Nerve Grafting and Matrix Modulation to Repair the Injured Rat Spinal Cord

Traumatic injury to the spinal cord (SCI) causes death of neurons, disruption of motor and sensory nerve fiber (axon) pathways and disruption of communication with the brain. One of the goals of our research is to promote axon regeneration to restore connectivity across the lesion site. To accomplish this we developed a peripheral nerve (PN) grafting technique where segments of sciatic nerve are either placed directly between the damaged ends of the spinal cord or are used to form a bridge across the lesion. There are several advantages to this approach compared to transplantation of other neural tissues; regenerating axons can be directed towards a specific target area, the number and source of regenerating axons is easily determined by tracing techniques, the graft can be used for electrophysiological experiments to measure functional recovery associated with axons in the graft, and it is possible to use an autologous nerve to reduce the possibility of graft rejection. In our lab we have performed both autologous (donor and recipient are the same animal) and heterologous (donor and recipient are different animals) grafts with comparable results. This approach has been used successfully in both acute and chronic injury situations. Regenerated axons that reach the distal end of the PN graft often fail to extend back into the spinal cord, so we use microinjections of chondroitinase to degrade inhibitory molecules associated with the scar tissue surrounding the area of SCI. At the same time we have found that providing exogenous growth and trophic molecules encourages longer distance axonal regrowth into the spinal cord. Several months after transplantation we perform a variety of anatomical, behavioral and electrophysiological tests to evaluate the recovery of function in our spinal cord injured animals. This experimental approach has been used successfully in several spinal cord injury models, at different levels of injury and in different species (mouse, rat and cat). Importantly, the peripheral nerve grafting approach is effective in promoting regeneration by acute and chronically injured neurons.

Traumatic injury to the spinal cord disrupts communication with the brain. To restore lost connectivity we utilize a peripheral nerve graft to provide a substratum for regenerating fibers in combination with neurotrophic factors and matrix-modulating enzymes to remove inhibitory molecules to promote long distance growth.

Traumatic injury to the spinal cord (SCI) causes death of neurons, disruption of motor and sensory nerve fiber (axon) pathways and disruption of ...

Biology

The Muscle Cuff Regenerative Peripheral Nerve Interface for the Amplification of Intact Peripheral Nerve Signals

Robotic exoskeletons have gained recent acclaim within the field of rehabilitative medicine as a promising modality for functional restoration for those individuals with extremity weakness. However, their use remains largely confined to research institutions, frequently operating as a means of static extremity support as motor detection methods remain unreliable. Peripheral nerve interfaces have arisen as a potential solution to this shortcoming; however, due to their inherently small amplitudes, these signals can be difficult to differentiate from background noise, lowering their overall motor detection accuracy. As current interfaces rely on abiotic materials, inherent material breakdown can occur alongside foreign body tissue reaction over time, further impacting their accuracy. The Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI) was designed to overcome these noted complications. Consisting of a segment of free muscle graft secured circumferentially to an intact peripheral nerve, the construct regenerates and becomes reinnervated by the contained nerve over time. In rats, this construct has demonstrated the ability to amplify a peripheral nerve's motor efferent action potentials up to 100 times the normal value through the generation of compound muscle action potentials (CMAPs). This signal amplification facilitates high accuracy detection of motor intent, potentially enabling reliable utilization of exoskeleton devices.

This manuscript provides an innovative method for developing a biologic peripheral nerve interface termed the Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI). This surgical construct can amplify its associated peripheral nerve's motor efferent signals to facilitate accurate detection of motor intent and the potential control of exoskeleton devices.

Robotic exoskeletons have gained recent acclaim within the field of rehabilitative medicine as a promising modality for functional restoration for those ...

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

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 cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...

Neuroscience

Functional and Physiological Methods of Evaluating Median Nerve Regeneration in the Rat

The main goal of this investigation is to show how to create and repair different types of median nerve (MN) lesions in the rat. Moreover, different methods of simulating postoperative physiotherapy are presented. Multiple standardized strategies are used to assess motor and sensory recovery using an MN model of peripheral nerve lesion and repair, thus permitting easy comparison of the results. Several options are included for providing a postoperative physiotherapy-like environment to rats that have undergone MN injuries. Finally, the paper provides a method to evaluate the recovery of the MN using several noninvasive tests (i.e., grasping test, pin prick test, ladder rung walking test, rope climbing test, and walking track analysis), and physiological measurements (infrared thermography, electroneuromyography, flexion strength evaluation, and flexor carpi radialis muscle weight determination). Hence, this model seems particularly appropriate to replicate a clinical scenario, facilitating extrapolation of results to the human species.
Although the sciatic nerve is the most studied nerve in peripheral nerve research, analysis of the rat MN presents various advantages. For example, there is a reduced incidence of joint contractures and automutilation of the affected limb in MN lesion studies. Furthermore, the MN is not covered by muscle masses, making its dissection easier than that of the sciatic nerve. In addition, MN recovery is observed sooner, because the MN is shorter than the sciatic nerve. Also, the MN has a parallel path to the ulnar nerve in the arm. Hence, the ulnar nerve can be easily used as the nerve graft for repairing MN injuries. Finally, the MN in rats is located in the forelimb, akin to the human upper limb; in humans, the upper limb is the site of most peripheral nerve lesions.

Presented is a protocol to produce different types of median nerve (MN) lesions and repair in the rat. Additionally, the protocol shows how to evaluate the functional recovery of the nerve using several noninvasive behavioral tests and physiological measurements.

The main goal of this investigation is to show how to create and repair different types of median nerve (MN) lesions in the rat. Moreover, different ...

Medicine

Implantation and Control of Wireless, Battery-free Systems for Peripheral Nerve Interfacing

Peripheral nerve interfaces are frequently used in experimental neuroscience and regenerative medicine for a wide variety of applications. Such interfaces can be sensors, actuators, or both. Traditional methods of peripheral nerve interfacing must either tether to an external system or rely on battery power that limits the time frame for operation. With recent developments of wireless, battery-free, and fully implantable peripheral nerve interfaces, a new class of devices can offer capabilities that match or exceed those of their wired or battery-powered precursors. This paper describes methods to (i) surgically implant and (ii) wirelessly power and control this system in adult rats. The sciatic and phrenic nerve models were selected as examples to highlight the versatility of this approach. The paper shows how the peripheral nerve interface can evoke compound muscle action potentials (CMAPs), deliver a therapeutic electrical stimulation protocol, and incorporate a conduit for the repair of peripheral nerve injury. Such devices offer expanded treatment options for single-dose or repeated dose therapeutic stimulation and can be adapted to a variety of nerve locations.

This is a protocol for the surgical implantation and operation of a wirelessly powered interface for peripheral nerves. We demonstrate the utility of this approach with examples from nerve stimulators placed on either the rat sciatic or phrenic nerve.

Peripheral nerve interfaces are frequently used in experimental neuroscience and regenerative medicine for a wide variety of applications. Such interfaces ...

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. This work describes the preparation of rat sciatic nerves for ex vivo neurophysiology, including buffer preparation, animal procedures, equipment setup and neurophysiological recording. This work provides an overview of the different types of experiments possible with this method. The outlined method aims to provide 6 h of stimulation and recording on extracted peripheral nerve tissue in tightly controlled conditions for optimal consistency in results. Results obtained using this method are A-fibre compound action potentials (CAP) with peak-to-peak amplitudes in the millivolt range over the entire duration of the experiment. CAP amplitudes and shapes are consistent and reliable, making them useful to test and compare new electrodes to existing models, or the effects of interventions on the tissue, such as the use of chemicals, surgical alterations, or neuromodulatory stimulation techniques. Both conventional commercially available cuff electrodes with platinum-iridium contacts and custom-made conductive elastomer electrodes were tested and gave similar results in terms of nerve stimulus strength-duration response.

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

This protocol describes the preparation of rat whole sciatic nerve tissue for ex vivo electrophysiological stimulation and recording in an environmentally-regulated, two-compartment, perfused saline bath.

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. ...

RPNI Surgery for Treating Postamputation Pain

Regenerative Peripheral Nerve Interface: Surgical Protocol for a Randomized Controlled Trial in Postamputation Pain

Surgical procedures, including nerve reconstruction and end-organ muscle reinnervation, have become more prominent in the prosthetic field over the past decade. Primarily developed to increase the functionality of prosthetic limbs, these surgical procedures have also been found to reduce postamputation neuropathic pain. Today, some of these procedures are performed more frequently for the management and prevention of postamputation pain than for prosthetic fitting, indicating a significant need for effective solutions to postamputation pain. One notable emerging procedure in this context is the Regenerative Peripheral Nerve Interface (RPNI). RPNI surgery involves an operative approach that entails splitting the nerve end longitudinally into its main fascicles and implanting these fascicles within&#160;free denervated and devascularized muscle grafts. The RPNI procedure takes a proactive stance in addressing freshly cut nerve endings, facilitating painful neuroma prevention and treatment by enabling the&#160;nerve to regenerate and innervate an end organ, i.e., the free muscle graft. Retrospective studies have shown RPNI&#39;s effectiveness in alleviating postamputation pain and preventing the formation of painful neuromas. The increasing frequency of utilization of this approach has also given rise to variations in the technique. This article aims to provide a step-by-step description of the RPNI procedure, which will serve as the standardized procedure employed in an international, randomized controlled trial (ClinicalTrials.gov, NCT05009394). In this trial, RPNI is compared to two other surgical procedures for postamputation pain management, specifically, Targeted Muscle Reinnervation (TMR) and neuroma excision coupled with intra-muscular transposition and burying.

Author Spotlight: Regenerative Peripheral Nerve Interface (RPNI) Surgery in Postamputation Pain Management

Here, we describe the surgical procedure to perform Regenerative Peripheral Nerve Interface (RPNI) surgery for treating postamputation neuropathic pain in the context of an international, randomized controlled trial (RCT) (ClinicalTrials.gov, NCT05009394). The RCT compares RPNI with two other surgical techniques, namely, Targeted Muscle Reinnervation (TMR) and neuroma excision combined with intra-muscular transposition.

Surgical procedures, including nerve reconstruction and end-organ muscle reinnervation, have become more prominent in the prosthetic field over the past ...

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

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