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 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.
Traumatic peripheral nerve injuries (PNIs) occur in the US with an annual incidence of approximately 200,000 per year1. Most patients who suffer PNIs are left with permanent functional impairments. At its worst, this may result in muscle paralysis and trigger treatment-refractory neuropathic pain so severe that patients are willing to undergo a limb amputation as treatment2. The biggest obstacle to improve PNI outcomes is that axon regeneration is too slow relative to the distances they must regrow. For example, an adult human axon grows at 1 mm/day but may have to regenerate over distances >1000 mm in the case of a lesion in a proximal limb.
In current clinical practice, ~50% of PNIs require surgical repair3. For successful nerve regeneration, axons must (i) grow across the lesion site (i.e., gap crossing) and then (ii) regenerate down the nerve pathway to reach an end-organ target (i.e., distal regrowth) (Figure 1). There are no FDA-approved drugs proven to accelerate nerve regeneration. The status quo of PNI clinical management has only changed incrementally over the past several decades and is limited to technical refinements to surgical methods such as distal motor nerve transfers to reduce the distance regenerating axons must travel4, or "off the shelf" synthetic nerve conduits for cases where the proximal nerve retracts and cannot be directly sutured back together5. However, there have been four randomized clinical trials on therapeutic electrical stimulation applied to nerves postoperatively, which were single-center studies led by Dr. K. Ming Chan at the University of Alberta that show significantly improved reinnervation of muscle6,7,8 or skin9. The foundational work for this electrical stimulation protocol was performed in rodents10,11, where it has been shown that electrical stimulation works specifically by enhancing gap crossing (Figure 1) but not distal regrowth12,13,14,15.
The surgical placement of transcutaneous wire electrodes used in all four electrical stimulation randomized clinical trials was necessary because its effects depend on the delivery of sufficient current to depolarize the neuron cell body at 20 Hz continuously for 1 h11. In clinical practice, this electrical stimulation protocol is not tolerable for most patients at the intensities required via surface-stimulating electrodes on the skin due to pain. There are non-trivial risks associated with running transcutaneous electrodes postoperatively, such as deep wound infection or accidental displacement of wires from the nerves during patient transport from the operating room (OR). Additionally, the high cost of OR time itself is a disincentive against attempting it in that setting rather than during acute postoperative recovery. A new class of wireless, battery-free, and fully implantable peripheral nerve interfaces is emerging to address this shortcoming of existing peripheral nerve interfaces.
This new class of wireless implantable electronic systems is poised to increase the ease and flexibility for electrical stimulation dosing and break down the barriers that preclude its broader clinical implementation. This paper describes methods to (i) surgically implant and (ii) wirelessly power and control this system in adult rat sciatic and phrenic nerve models. It shows how the peripheral nerve interface can evoke CMAPs, deliver a therapeutic electrical stimulation protocol, and even act as a conduit for the repair of peripheral nerves. The protocols here can be adapted for other variants of this technology that can deliver light pulses for optogenetic mediated neuromodulation16, controlled drug release17, or repeated bouts of electrical stimulation over time18,19.
All procedures described in this protocol are carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of Northwestern University. This protocol follows the animal care guidelines of Northwestern University's Center for Comparative Medicine and IACUC. It is necessary to consult with IACUC when adapting the protocols.
1. Wireless electronic stimulator fabrication (Figure 2)
2. Device preparation for implantation
3. Surgical procedure of rat right sciatic nerve implantation of wireless, battery-free peripheral nerve interface for electrical stimulation (Figure 3)
NOTE: Maintain sterile conditions. Perform surgeries within the designated surgical area of an animal procedure room. The surgeon will don a facemask, coat, cap, and sterile gloves during surgery. If more than one surgery is performed, change sterile gloves between animals and use clean, sterile surgical instruments for each surgery. Sterilize tools between surgeries by heat sterilization (autoclave or glass bead sterilizer). Use adult Sprague-Dawley rats that weighing 200-250 g.
4. Surgical procedure of rat left phrenic nerve implantation of wireless stimulators (Figure 5A)
NOTE: Maintain sterile conditions, as in section 3. Use adult Sprague-Dawley rats weighing 200-250 g. Sterilize all surgical tools before use.
5. Wireless delivery of therapeutic electrical stimulation
6. Euthanasia
In the sciatic nerve injury model, the implant is placed around the right sciatic nerve prior to end-to-end repair of the tibial nerve branch (Figure 3, Figure 4A, and Figure 7A). A 30 G concentric needle electrode is placed in the right tibialis anterior muscle to define the stimulus parameters needed for maximal intensity electrical stimulation. These experiments include elevating the stimulation intensity until the response magnitude plateaus at the maximum. As the tibialis anterior is innervated by the fibular branch of the sciatic nerve, it is spared in the tibial nerve transection injury. Thus, recording from tibialis anterior enables continuous monitoring of the electrical stimulation treatment.
For a single-stimulus pulse delivered by a wire electrode to the right sciatic nerve (5 mA, 0.02 ms), a maximal CMAP response is elicited with a 5.4 mV negative peak amplitude recorded on ipsilateral tibialis anterior (Figure 7B; black trace). For a comparable stimulus pulse delivered by the wireless, battery-free implant, a comparable CMAP response is elicited with a 4.6 mV negative peak amplitude (Figure 7B; orange trace). This is consistent with a recent report that wireless nerve stimulation achieves on average 88% of the CMAP from wire-based nerve stimulation21, well above the threshold required for therapeutic effects in clinical studies6,7,8,9. In the example shown, the longer latency of the wireless stimulator vs. wired stimulator was due to its greater distance from the recorded muscle.
In the phrenic nerve model, the implant is placed around the right phrenic nerve prior to transection (Figure 5). To define the stimulus parameters needed for maximal intensity electrical stimulation, a 30 G concentric needle electrode is placed subcutaneously at the right (ipsilateral) anterior costal margin to record from the right hemidiaphragm. The experiments involve elevating the stimulation voltage till the response magnitude plateaus at its maximum. As the phrenic nerve can be challenging to isolate from surrounding neurovascular structures, its identity can be confirmed by evoking a twitch response (Figure 6; orange trace). The specificity of stimulation can be further verified by transection of the phrenic nerve distal to the nerve electrode cuff with subsequent abolishment of the twitch response (Figure 6; black trace).
Repetitive, low-frequency electrical stimulation therapy can be delivered to the sciatic nerve for 1 h using an established protocol that enhances axon regeneration (6,7,8,9,10,11; Figure 8). The cuff interface of the wireless implant was placed on the right sciatic nerve, and the 30 G concentric needle electrode was placed on the right tibialis anterior muscle to monitor the treatment. Figure 8A shows four sequential spikes in the recorded electromyography at the beginning (0 min) of the 1 h 20 Hz electrical stimulation. Figure 8B shows four other spikes recorded at the 40 min of the 1 h electrical stimulation with a slight decrease in peak amplitude, which is consistent with the fatigue pattern noted with wire-based electrical stimulation therapy15,21.
The degree of peripheral nerve regeneration can be assessed using retrograde tracers applied distally to the nerve lesion site. Because peripheral axons sprout multiple collateral sprouts, retrograde tracing and counts of the motor neuron soma in the spinal cord allow a more accurate assessment of the number of regenerating neurons than counting regenerating axons within the nerve itself31. To demonstrate this, the sciatic nerve trunk was transected by a crush injury. After 3 weeks of recovery, two different fluorescent retrograde dyes were administered on two branches of the sciatic nerve: fibular nerve (green) and tibial nerve (red), respectively (Figure 9A). Figure 9B-D show lit-up subgroups of lower motoneurons in the lumbar spinal cord anterior horn that form either the tibial nerve (Figure 9B) or the fibular nerve (Figure 9C). The overlay image shows two distinct columns of labeled neurons in the anterior horn of the spinal cord, which can be quantified in terms of spatial distribution and the count of motor neurons that have regenerated an axon distal to the lesion site (Figure 9D).
Figure 1: Nerve regeneration model. (A) Gap crossing occurs early after nerve repair when axons grow from proximal to distal nerve end after repair. (B) The duration of distal regrowth is related to the distance to the target end-organ (e.g., skin, muscle) and the rate of axon regrowth. Most therapies for improving nerve repair target one or both of these processes. Please click here to view a larger version of this figure.
Figure 2: Illustration of a wireless electronic stimulator fabrication. Left, detailed layers of the structure of the device, including a circular radio frequency power harvester coil, a stretchable extension electrode, and a nerve cuff wrapping around a nerve of interest. Right, a simplified illustration showing three parts of the device. Abbreviations: PLGA = poly(lactic-co-glycolic acid); b-DCPU = bioresorbable dynamic covalent polyurethane. Please click here to view a larger version of this figure.
Figure 3: Implantation of wireless, battery-free nerve interface in the rat sciatic nerve model. (A) The illustration depicts a fully implantable system in the right sciatic nerve of a rat. (B) The top panel shows an electrode interface positioned on the sciatic nerve just proximal to the end-to-end repair of the right tibial nerve. The bottom panel shows an electrode interface with an extended nerve cuff bridging gap repair between the proximal end and the distal nerve stump. Abbreviation: PLGA = poly(lactic-co-glycolic acid). Please click here to view a larger version of this figure.
Figure 4: Sciatic nerve implantation procedure. (A) Incision on the skin, subcutaneous connective tissue, and the gluteal muscle to expose the hamstring. (B) Isolated sciatic nerve (black arrow). (C) Device post-implantation with nerve cuff, wires (white asterisk), and implant visible (star). (D) Closure of the connective tissue by suture. (E) Closure of the incision by wound clips. (F) Wireless electrical stimulation generated by a coil above the skin. Please click here to view a larger version of this figure.
Figure 5: Phrenic nerve implantation procedure. (A) Ventral view of the neck in the supine position. (B) Incision on the skin and subcutaneous connective tissue to expose sternohyoid muscle. (C) Dissecting through the potential space between the omohyoid muscle and the sternocleidomastoid muscle. (D) Phrenic nerve (arrow), isolated from the brachial plexus. (E) Diaphragmatic electromyographic confirmation of the phrenic nerve. Black arrow, recording electrode. Red arrow, stimulators. (F) Implantation. (G) Closure of the skin with deep dermal stitches. Please click here to view a larger version of this figure.
Figure 6: Confirmation of complete phrenic nerve transection injury by evoked compound muscle action potentials from the diaphragm. Before phrenic nerve transection (ORANGE), electrical stimulation of the phrenic nerve evoked compound muscle action potentials on the ipsilateral diaphragm, which was abolished by phrenic nerve transection (BLACK). Please click here to view a larger version of this figure.
Figure 7: Representative nerve conduction studies comparing wireless to wire-based electrical stimulation. (A) Illustration of wireless (BLACK) and wired (ORANGE) devices placements on the sciatic nerve. The recording electrode was placed in the tibialis anterior. (B) Compound muscle action potentials evoked by wired implant (ORANGE) vs. wireless implant (BLACK). Please click here to view a larger version of this figure.
Figure 8: EMG recording from TA muscle with 20 Hz repetitive electrical stimulation for 1 h from implants. (A) Trace of EMG at min 1 of e-stim. (B) Trace of EMG at min 40 of e-stim. Abbreviations: EMG = electromyography; TA = tibialis anterior; e-stim = electrical stimulation; min = minute. Please click here to view a larger version of this figure.
Figure 9: Representative images of sciatic nerve regeneration. (A) Illustration of sciatic nerve injury and fluorescent retrograde labeling. The sciatic nerve axons were transected by crush injury. After 3 weeks of recovery, its distal branches-the fibular nerve (in green) and tibial nerve (in red)-were retrogradely labeled. (B-D) Images of a lumbar spinal cord showing neuronal soma within the ipsilesional anterior horn. Scale bars = 30 µm. Please click here to view a larger version of this figure.
This paper describes the steps in the surgical implantation and operation of a wireless, battery-free, and fully implantable peripheral nerve interfaces in the rat sciatic and phrenic nerve model. We demonstrate how this novel class of biomedical implants can be used to deliver a therapeutic electrical stimulation paradigm shown to enhance axon regeneration in preclinical and clinical studies (for review, see22). This protocol is uncomplicated and can be extrapolated to smaller animal models, such as mice21, as well as other wireless, battery-free, and fully implantable devices with functionality that includes optoelectronic and microfluidic peripheral nerve interfaces18,23,24,25,26,27,28,29,30. Also demonstrated is the approach using the rodent sciatic nerve, which is the most common experimental model31.
The versatility of this approach has been shown when it is adapted to interface with the phrenic nerve, which is rarely employed as a model of peripheral nerve injury32, perhaps because it is a vastly under-recognized clinical issue33,34,35. Phrenic nerve injury diagnosis and rehabilitation have become an important issue during the COVID-19 pandemic36,37,38. It is currently unknown whether regeneration of phrenic axons and recovery from diaphragm paralysis can be augmented by this brief, low-frequency electrical stimulation paradigm. However, phrenic nerve electrical stimulation for diaphragm muscle pacing is an established option for respiratory failure in patients with tetraplegia from high cervical spinal cord injury39,40,41,42,43. Other indications are being explored, including ventilator weaning after critical illness44.
Several critical steps should be emphasized to ensure good operation of the implanted system. First, it is important to avoid applying too much force on the thin electronic components of the devices when handling them to prevent lead de-insulation, kinking, or breakage. Next, it is important to accurately mark the location of the radio frequency power harvester coil on the overlying skin. Third, careful alignment of the transmission coil of the external radio frequency power supply over the power harvester coil of the implanted device with a gooseneck clamp allows for stable operation. Finally, to confirm electrical stimulation in addition to visual observation of muscle twitching, periodic neurophysiological monitoring is recommended. In the case of the more complex anatomy of the phrenic nerve in the neck, electrophysiological confirmation helps demonstrate that the correct nerve has been isolated (Figure 6).
Besides the wireless, battery-free electrical stimulators shown in this paper18,19,21, many other devices potentially share the same procedures. For instance, because electrodes designed to implant to the glossopharyngeal and vagus nerves to chronically record signals from sympathetic and parasympathetic nervous systems30,45,46 share a similar surgical area with the phrenic nerve, this protocol can be adapted for their implantation. Wireless long-term biocompatible stimulators for peripheral nerves, such as ReStore, are great tools to remain in place and stimulate nerves as needed25,47,48,49,50. Relevant multi-channel wireless recording implants have also been reported51. Overall, we believe these surgical and electrical stimulation protocols can be adapted as a standard for all wireless peripheral nerve interfacing related to electrical stimulation or recording.
This work used the NUFAB facility of Northwestern University's NUANCE Center, which has received support from the SHyNE Resource (NSF ECCS-1542205), the IIN, and Northwestern's MRSEC program (NSF DMR-1720139). This work made use of the MatCI Facility supported by the MRSEC program of the National Science Foundation (DMR-1720139) at the Materials Research Center of Northwestern University. C.K.F acknowledges support from Eunice Kennedy Shriver Institute of Child Health and Human Development of the NIH (grant no. R03HD101090) and the American Neuromuscular Foundation (Development Grant). Y.H. acknowledges support from NSF (grant no. CMMI1635443). This work was supported by the Querrey Simpson Institute for Bioelectronics at Northwestern University.
Name | Company | Catalog Number | Comments |
Amplifier | Electronics & Innovation | 201L | |
Arbitrary Waveform Generator | RIGOL | DG1032Z | 30 MHz, 2 Channel, 200 MS/s, 14bit Resolution, 8 Mpts |
Bupivacaine | Pfizer | 655317 | Marcaine, 0.5% |
Copper/polyimide/copper | Pyralux | AP8535R | 18 µm thick top and bottom copper, 75 µm thick polyimide |
EMG recording device | Natus | Nicolet VikingQuest | |
EPOXY MARINE | Loctite | ||
Isoflurane, USP | Butler Schein Animal Health | 1040603 | ISOTHESIA |
Meloxicam | covetrus | 5mg/ml | |
Needle electrodes | Technomed USA Inc. | TE/B50600- 001 | |
PDMS (Silicone Elastomer Kit) | DOW | SYLGARDâ„¢ 184 | |
ProtoLaser U4 | LPKF | U4 | |
Puralube Vet Ointment Sterile Ocular Lubricant | Puralube | 83592 | |
Waveform generator | Agilent Technologies | Agilent 33250A |
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