Interfacing peripheral nerve with macro-sieve electrodes following spinal cord injury

Abstract

Macro-sieve electrodes were implanted in the sciatic nerve of five adult male Lewis rats following spinal cord injury to assess the ability of the macro-sieve electrode to interface regenerated peripheral nerve fibers post-spinal cord injury. Each spinal cord injury was performed via right lateral hemisection of the cord at the T_9–10 site. Five months post-implantation, the ability of the macro-sieve electrode to interface the regenerated nerve was assessed by stimulating through the macro-sieve electrode and recording both electromyography signals and evoked muscle force from distal musculature. Electromyography measurements were recorded from the tibialis anterior and gastrocnemius muscles, while evoked muscle force measurements were recorded from the tibialis anterior, extensor digitorum longus, and gastrocnemius muscles. The macro-sieve electrode and regenerated sciatic nerve were then explanted for histological evaluation. Successful sciatic nerve regeneration across the macro-sieve electrode interface following spinal cord injury was seen in all five animals. Recorded electromyography signals and muscle force recordings obtained through macro-sieve electrode stimulation confirm the ability of the macro-sieve electrode to successfully recruit distal musculature in this injury model. Taken together, these results demonstrate the macro-sieve electrode as a viable interface for peripheral nerve stimulation in the context of spinal cord injury.

Introduction

Spinal cord injury (SCI) is a debilitating condition that is detrimental to the wellbeing and productivity of affected individuals. In the United States alone, 282,000 individuals are estimated to be living with a SCI, with ~17,000 new cases occurring each year, primarily in young adults (National Spinal Cord Injury Statistical Center, 2016). Injury to the mammalian spinal cord causes neuron death at lesion site with local loss of anterior horn cells. This ultimately results in injury-dependent losses to motor function distal to the site of injury. While axons in the peripheral nervous system (PNS) are capable of robust regeneration post-injury, axons in the central nervous system (CNS) are not and thus loss of function due to SCI is typically permanent (Huebner and Strittmatter, 2009). One promising approach to restoring motor function to SCI patients is the use of peripheral nerve interfaces (PNIs). A PNI is a micro-electrode array used to stimulate or record from a peripheral nerve, in this case one distal to the spinal cord lesion. Many types of PNIs are being developed as potential modalities for neuromuscular control and delivery of functional electrical stimulation. In this paper, we discuss the applicability of the macro-sieve electrode (MSE) as a potential target for restoring motor function following SCI

Interfacing Peripheral Nerve in the Context of SCI

PNIs are broadly classified into three types—extraneural electrodes, penetrating intraneural electrodes, and regenerative electrodes. Extraneural electrodes, such as the cuff electrode (Veraart et al., 1993) or the flat interface nerve electrode (Tyler and Durand, 2002), are minimally invasive but achieve only limited selective muscular recruitment. Penetrating intraneural electrodes, such as the Utah slant electrode array (Branner et al., 2004) and the transverse intrafascicular multichannel electrode (Boretius et al., 2010), are inserted directly into the target nerve to gain selective intrafascicular control, but suffer from complications such as the breakage of the tines and fibrous encapsulation (Gasson et al., 2004; Christensen et al., 2014). Regenerative electrodes, such as the MSE, interface peripheral nerve that has regenerated across the electrode interface from two surgically opposed nerve stumps (Thompson et al., 2016). By interfacing directly with this regenerated nerve, the MSE is able to provide selective control while avoiding issues such as breakage and scarring (MacEwan et al., 2016).

Direct nerve integration with any regenerative electrode, however, requires transection of the nerve of interest and depends entirely on the regenerative capacity of the nerve – two complicating factors which determine the applicability of regenerative electrodes (Lago et al., 2007). Furthermore, implantation of an MSE subjects the PNS to a second injury following the initial CNS lesion, which intuitively may affect the peripheral nerve's regenerative capacity, as peripheral nerve axons distal to the SCI lesion have altered structural morphology (Redondo-Castro and Navarro, 2013). Our clinical experience and a growing body of literature demonstrating clinical recovery using peripheral nerve transfers distal to the site of injury suggests that regenerative electrodes such as the MSE may provide an interface that promotes a more widespread use of caudal spinal segments in SCI patients (Ray et al., 2016). We sought to investigate whether a MSE implanted post-SCI could still be used to selectively recruit distal musculature.

Capabilities of the MSE

Details of the MSE design and fabrication have been described previously (MacEwan et al., 2016). Briefly, the MSE is a high-transparency regenerative sieve electrode featuring nine large transit zones, each with an area of approximately 0.285 mm². These transit zones are bordered by eight radial spokes and a central ring which are metallized with Pt-Ir to yield four central and four peripheral active electrode sites (Figure 1A). Each MSE assembly also features silicone nerve guidance conduits which project 3 mm from each face and enable the MSE to be secured to the epineurium during implantation.

Figure 1.

Macrosieve electrode.

(A) Optical micrograph of MSE (without connector assembly). (B) MSE with connector assembly implanted in transected sciatic nerve 5 mm proximal to the point of trifurcation. MSE: Macro-sieve electrode.

Several characteristics of the MSE make it especially attractive in the context of SCI compared to other PNIs. The MSE can recruit highly selective groups of regenerated nerve fibers in uninjured animals (MacEwan et al., 2016), meaning that selective motor control can potentially be provided to multiple muscle groups affected by SCI through a single MSE. Specifically, in uninjured animals, the MSE has been able to recruit up to 50% of maximal evoked muscle twitch force using only monopolar stimulation paradigms. Additionally, as the MSE is not implanted via penetration, it does not experience the mechanical or foreign-body response complications associated with intraneural electrodes (Christensen et al., 2014), furthering its applicability as a long-term PNI for use in SCI. However, as with any regenerative sieve electrode, the ability of the MSE to selectively interface the nerve relies on robust regeneration across its interface, and thus potential hurdles to this regeneration must be carefully evaluated. It is for this reason that we have sought to investigate whether there are any negative downstream effects of SCI on regeneration across the MSE interface.

Evaluation of the MSE in SCI

MSE were implanted in the sciatic nerve of five adult male Lewis rats following SCI to assess the ability of the MSE to interface regenerated peripheral nerve fibers post-SCI. Each SCI was performed via right lateral hemisection of the cord at the T_9–10 site. Two weeks post-SCI, an MSE was implanted in the right sciatic nerve. For this procedure, the sciatic nerve was transected 5 mm proximal to the point of trifurcation. An MSE was placed in the transected nerve gap and the proximal and distal nerve stumps were sutured into either end of the silicone nerve guidance conduit by the epineurium (Figure 1B).

Five months post-implantation, the ability of the MSE to interface the regenerated nerve was assessed by stimulating through the MSE and recording both electromyography (EMG) signals and evoked muscle force measurements from distal musculature. An identical set of stimuli was used to stimulate the regenerated nerve for both EMG and muscle force recordings. Individual stimuli consisted of a biphasic, square, symmetrical pulse of current between 100 μA and 500 μA delivered over 1 ms (i.e., 100 μA for 0.5 ms then –100 μA for 0.5 ms). Stimuli were delivered cathodically through the implanted MSE using a MS16 stimulus isolator (Tucker-Davis Technologies, Inc., Alachua, FL, USA) connected to a desktop PC via optical cable.

For the EMG measurements, recording needle electrodes were placed in the tibialis anterior (TA) and gastrocnemius (G) muscles. Additional counter and reference needle electrodes were placed subcutaneously in the lower back of the animal. Recorded EMG signals were routed through a RA16LI-D 16-channel differential recording head stage and amplified using a RA16PA 16-channel medusa preamp (Tucker-Davis Technologies, Inc., Alachua, FL, USA) before being sent to a desktop PC via optical cable using custom OpenEx data acquisition software (Tucker-Davis Technologies, Inc., Alachua, FL, USA).

For muscle force recordings, the anterolateral aspect of the right hind limb was exposed to facilitate access to the tendons of the TA, extensor digitorum longus (EDL), and G muscles. Distal tendons of the target muscles were cut and secured to separate stainless-steel S-hooks using 5-0 nylon suture. The right leg was then immobilized at the femoral condyles by use of a C-clamp. The stainless-steel S-hook was then connected to a 5 N thin-film load cell force sensor (Strain Measurement Devices, Inc., Meriden, CT, USA). Evoked muscle forces for each the TA, EDL, and G muscles were transduced individually via the force sensor and recorded on a desktop PC using the previously described hardware and software.

All channels in each animal reached an EMG response plateau atstimulation values of 150–200 μA (Figure 2), consistent with data from uninjured animals (MacEwan et al., 2016). A similar plateau was obtained for evoked muscle force recordings, in which recorded muscle force normalized by the maximum muscle force for each muscle also reached a maximum at stimulation values of 150–200 μA. When recording evoked muscle response due to electrical stimulation, a sigmoidal curve typically appears as smaller currents do not cause the motor fibers to reach threshold, while higher currents recruit increasingly more motor fibers until all fibers are recruited and a plateau is reached. However, due to the absence of lower stimulation values and the use of comparatively large current step sizes in this experiment, it is difficult to visualize the expected sigmoidal recruitment curve.

Figure 2.

Population tibialis anterior (TA) and gastrocnemius (G) EMG data for stimulation through each macro-sieve electrode (MSE) site in each animal.

Red lines represent the mean signal response across all MSE sites in each animal, while the blue upper and lower bounds of the boxes represent the 75^th and ²⁵th percentile respectively. Maxima and minima are marked using black whiskers.

Successful sciatic nerve regeneration across the MSE interface following spinal cord injury was visually observed in all five rats. EMG and muscle force recordings obtained following stimulation through the MSE confirm the ability of the MSE to successfully recruit distal musculature in this injury model. Taken together, these results demonstrate that the MSE is a viable interface for providing functional neuromuscular stimulation following SCI.

Conclusion

The PNS offers an attractive biological target for neuroprosthetic devices aimed at restoring motor function following SCI. Unfortunately, microelectrode devices developed to date have not been able to achieve a stable, chronic, high-specificity interface with peripheral nerve tissue required for high resolution muscle activation and motor control. MSEs represent a novel approach to achieving a chronic, stable, high-specificity interface with peripheral nerve tissue for the purpose of muscle activation and motor control.

The present study represents the first instance of regenerative sieve electrodes being applied as a means of interfacing peripheral nerve tissue and providing motor activation in the context of SCI. Further work is needed to determine the clinical potential of MSEs in the context of SCI.

Additional file ^{(276.6KB, pdf)} : Open peer review report 1, 2.

Additional file:

Open peer review report 1 on “Interfacing peripheral nerve with macro-sieve electrodes following spinal cord injury”.