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Optical Stimulation for Restoration of Motor Function After Spinal Cord Injury

      Abstract

      Spinal cord injury can be defined as a loss of communication between the brain and the body due to disrupted pathways within the spinal cord. Although many promising molecular strategies have emerged to reduce secondary injury and promote axonal regrowth, there is still no effective cure, and recovery of function remains limited. Functional electrical stimulation (FES) represents a strategy developed to restore motor function without the need for regenerating severed spinal pathways. Despite its technological success, however, FES has not been widely integrated into the lives of spinal cord injury survivors. In this review, we briefly discuss the limitations of existing FES technologies. Additionally, we discuss how optogenetics, a rapidly evolving technique used primarily to investigate select neuronal populations within the brain, may eventually be used to replace FES as a form of therapy for functional restoration after spinal cord injury.

      Abbreviations and Acronyms:

      ChR-2 (channelrhodopsin-2), FES (functional electrical stimulation), SCI (spinal cord injury)
      CME Activity
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      Credit Statement: Mayo Clinic College of Medicine designates this journal-based CME activity for a maximum of 1.0 AMA PRA Category 1 Credit(s).TM Physicians should claim only the credit commensurate with the extent of their participation in the activity.
      Learning Objectives: On completion of this article, you should be able to (1) summarize the current state of optogenetic technology for restoring motor function in animal models, (2) describe current investigational uses of optogenetics, (3) identify limitations facing translation of optogenetics to human therapeutics.
      Disclosures: As a provider accredited by ACCME, Mayo Clinic College of Medicine (Mayo School of Continuous Professional Development) must ensure balance, independence, objectivity, and scientific rigor in its educational activities. Course Director(s), Planning Committee members, Faculty, and all others who are in a position to control the content of this educational activity are required to disclose all relevant financial relationships with any commercial interest related to the subject matter of the educational activity. Safeguards against commercial bias have been put in place. Faculty also will disclose any off-label and/or investigational use of pharmaceuticals or instruments discussed in their presentation. Disclosure of this information will be published in course materials so that those participants in the activity may formulate their own judgments regarding the presentation.
      In their editorial and administrative roles, William L. Lanier, Jr, MD, Terry L. Jopke, Kimberly D. Sankey, and Nicki M. Smith, MPA, have control of the content of this program but have no relevant financial relationship(s) with industry.
      Dr Lujan has intellectual property licensed to Boston Scientific Corporation. The authors declare that this review was composed in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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      Date of Release: 2/1/2015
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      Questions? Contact [email protected] .
      Despite efforts to elucidate the pathophysiology of spinal cord injury (SCI) in the past few decades, the search for a cure continues.
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      An alternative to molecular manipulations is to activate remaining neuromuscular components, which, despite the loss of descending input, can still be activated via external stimuli. Historically, the most common form of stimuli has been electricity. Namely, functional electrical stimulation (FES) has been successfully used to restore breathing,
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      Presently, FES systems can restore lost function, but they have a narrow scope of application and generally only restore one previously lost function at a time. For example, phrenic pacing has allowed individuals with high cervical injuries and intact phrenic nerves to successfully wean from mechanical ventilation, leading to increased survival rates and improved quality of life.
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      Additionally, Parastep (Sigmedics, Inc), a commercially available device that relies on surface stimulation of the quadriceps, gluteal muscles, and peroneal nerves, permits individuals with lower SCI to ambulate for distances of more than a quarter of a mile.
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      and muscle fatigue
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      have led to a limited integration of FES systems into the daily lives of SCI survivors.
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      Optogenetics, a novel stimulation modality that uses light to either excite or inhibit genetically modified neurons, has the potential to overcome some of the limitations facing current FES strategies.
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      Optogenetic dissection reveals multiple rhythmogenic modules underlying locomotion.

      Optogenetics

      Optogenetics is a rapidly evolving technique originally developed to study neural activity in select neuronal populations.
      • Deisseroth K.
      Optogenetics.
      The genetic material of specific cell populations is modified via viral vectors to express a transmembrane protein reactive to light (opsins). These transmembrane proteins undergo a conformational change when light of a specific wavelength (390-700 nm) is applied directly to the cells, resulting in selective ionic current flow across the cell membrane. In turn, positively charged (cations) or negatively charged (anions) ionic movement will lead to cell depolarization or hyperpolarization, respectively. Therefore, specific viral vectors can be chosen and modified to transduce specific neuronal populations, allowing for selective modulation with light. Excitatory responses can be achieved by activating channelrhodopsin-2 (ChR-2) cation channels (responsive to 470-nm wavelength blue light), which allow entry of positively charged sodium and calcium ions into the cell (Figure 1, A).
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      Optogenetic control of targeted peripheral axons in freely moving animals.
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      Millisecond-timescale, genetically targeted optical control of neural activity.
      In contrast, inhibitory responses can be evoked by activating halorhodopsin, a transmembrane ion pump, using 580-nm yellow light, which facilitates the movement of negatively charged chloride ions (Figure 1, B).
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      Multimodal fast optical interrogation of neural circuitry.
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      Optogenetic dissection reveals multiple rhythmogenic modules underlying locomotion.
      Figure thumbnail gr1
      Figure 1Mechanisms of neuromodulation via optogenetics. A, Application of blue light (470-nm wavelength) leads to a conformational change of the transmembrane ion channel protein channelrhodopsin (ChR2), allowing a flow of positively charged ions into the cytoplasm, ultimately leading to neuron depolarization. B, Yellow light application (580-nm wavelength) changes the conformation of the transmembrane ion pump protein halorhodopsin (HR), allowing negatively charged ions to move into the cytoplasm, leading to neuron hyperpolarization. C, Schematic comparing the nonspecific activation characteristic of electrical activation, leading to both desired and undesired effects, and optical activation of only targeted neurons, leading to only desired effects. Ca2+ = calcium ion; Cl = chloride ion; H+ = hydrogen ion; K+ = potassium ion; Na+ = sodium ion.
      Adapted by permission from Macmillan Publishers Ltd: Nat Rev Neurosci,
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      copyright 2007.
      The use of optogenetics has previously focused on characterization of neuronal mechanisms of excitation and inhibition within the brain.
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      • Wang L.-P.
      • Brauner M.
      • et al.
      Multimodal fast optical interrogation of neural circuitry.
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      • Wang E.H.
      • Woodson W.J.
      • et al.
      Optogenetic neuronal stimulation promotes functional recovery after stroke.
      • Tyan L.
      • Chamberland S.
      • Magnin E.
      • et al.
      Dendritic inhibition provided by interneuron-specific cells controls the firing rate and timing of the hippocampal feedback inhibitory circuitry.
      • Cho J.H.
      • Deisseroth K.
      • Bolshakov V.Y.
      Synaptic encoding of fear extinction in mPFC-amygdala circuits.
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      • Carels V.M.
      • Deisseroth K.
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      Optogenetic activation of an inhibitory network enhances feedforward functional connectivity in auditory cortex.
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      • Frechette E.S.
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      Closed-loop optogenetic control of thalamus as a tool for interrupting seizures after cortical injury.
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      Electrical stimulation of the conus medullaris to control the bladder in the paraplegic patient: a 10-year review.
      However, increased interest in translational applications of optogenetics technology has resulted in the pursuit of novel clinical avenues for restoration of vision, seizure control, and treatment of cardiac arrhythmias.
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      Light offers clear advantages for modulating neuronal behavior. Specifically, optical stimulation can provide real-time, selective control of cellular activity.
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      • Zhang F.
      • Bamberg E.
      • Nagel G.
      • Deisseroth K.
      Millisecond-timescale, genetically targeted optical control of neural activity.
      Additionally, efforts to expand the toolbox for controlling neurons via light have led to an increased variety of ChR-2s that are altered to respond to various light wavelengths with enhanced ion channel kinetics and selectivity.
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      Color-tuned channelrhodopsins for multiwavelength optogenetics.
      More recent efforts have led to the first light-gated chloride channel, engineered from the ChR-2 transmembrane family of proteins, which is designed to decrease the latency between light activation and cell inhibition that is observed with halorhodopsin ion pumps.
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      Conversion of channelrhodopsin into a light-gated chloride channel.
      Furthermore, optical control of muscle function has been achieved by controlling murine stem cells previously engineered to express ChR-2, followed by implantation distal to a nerve ligation in an attempt to establish a possible regenerative medicine therapeutic intervention.
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      Moreover, the combination of genes that express ChR-2 with genes that express the light-generating protein luciferase revealed that it is possible to activate neurons by exogenous application of the luciferase substrate, leading to cell luminescence and light-driven autoactivation in vitro.
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      Finally, computational modeling evidence has illustrated that optogenetic activation of axons follows a physiologic, small- to large-diameter axon recruitment order,
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      Theoretical principles underlying optical stimulation of myelinated axons expressing channelrhodopsin-2.
      which could prove invaluable for restoring motor function following SCI.

      Restoration of Motor Function After SCI Via Optical Stimulation

      Applications of optogenetic technology for restoring function after SCI are already under way in small animal models. In fact, optogenetics has recently been used to dissect select spinal cord circuitry responsible for evoking both rhythmic and stimulation-triggered limb movements.
      • Hägglund M.
      • Borgius L.
      • Dougherty K.J.
      • Kiehn O.
      Activation of groups of excitatory neurons in the mammalian spinal cord or hindbrain evokes locomotion.
      • Hägglund M.
      • Dougherty K.J.
      • Borgius L.
      • Itohara S.
      • Iwasato T.
      • Kiehn O.
      Optogenetic dissection reveals multiple rhythmogenic modules underlying locomotion.
      • Llewellyn M.E.
      • Thompson K.R.
      • Deisseroth K.
      • Delp S.L.
      Orderly recruitment of motor units under optical control in vivo.
      Specifically, Towne et al
      • Towne C.
      • Montgomery K.L.
      • Iyer S.M.
      • Deisseroth K.
      • Delp S.L.
      Optogenetic control of targeted peripheral axons in freely moving animals.
      reported the ability of using optical stimulation to selectively activate hind limb muscles in a rodent model of SCI using retrograde transduction of motor neurons with ChR-2 via intramuscular inoculation with an adeno-associated virus. Similarly, Alilain et al
      • Alilain W.J.
      • Li X.
      • Horn K.P.
      • et al.
      Light-induced rescue of breathing after spinal cord injury.
      found that it is possible to restore motor activity in the diaphragm muscle of rodents that sustained a cervical SCI using optical stimulation of the spinal cord at cervical vertebra C3 through C6. Additionally, Hägglund et al
      • Hägglund M.
      • Dougherty K.J.
      • Borgius L.
      • Itohara S.
      • Iwasato T.
      • Kiehn O.
      Optogenetic dissection reveals multiple rhythmogenic modules underlying locomotion.
      reported rhythmic activation of selective muscles necessary for locomotion using optical stimulation in a transgenic mouse line expressing ChR-2 channels in spinal interneurons.
      The continued development of optogenetic technology promises to overcome several limitations of electrical stimulation techniques for restoring motor function after SCI. First, optical stimulation allows selective muscle activation and fine motor control because of increased specificity associated with viral transduction of select motor neurons,
      • Llewellyn M.E.
      • Thompson K.R.
      • Deisseroth K.
      • Delp S.L.
      Orderly recruitment of motor units under optical control in vivo.
      as well as the theoretical possibility of direct transduction and control of skeletal muscle itself.
      • Bruegmann T.
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      • Hesse M.
      • et al.
      Optogenetic control of heart muscle in vitro and in vivo.
      Second, optogenetics may restore function in a more physiologic manner, particularly for functions that involve complex patterns of excitation and inhibition of different neuronal populations. An example is micturition, which requires activation of parasympathetic neural circuitry to initiate bladder emptying and simultaneous inhibition of sphincter contraction. Although electrical stimulation can be used to achieve bladder emptying,
      • Grill W.M.
      • Bhadra N.
      • Wang B.
      Bladder and urethral pressures evoked by microstimulation of the sacral spinal cord in cats.
      • Yoo P.B.
      • Klein S.M.
      • Grafstein N.H.
      • et al.
      Pudendal nerve stimulation evokes reflex bladder contractions in persons with chronic spinal cord injury.
      • Liske H.
      • Towne C.
      • Anikeeva P.
      • et al.
      Optical inhibition of motor nerve and muscle activity in vivo.
      it occurs in a suboptimal tetanic fashion. Third, muscle fatigue associated with existing electrical stimulation technologies
      • Fisher L.E.
      • Miller M.E.
      • Nogan S.J.
      • et al.
      Preliminary evaluation of a neural prosthesis for standing after spinal cord injury with four contact nerve-cuff electrodes for quadriceps stimulation.
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      Recruitment properties of intramuscular and nerve-trunk stimulating electrodes.
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      Extracellular voltage profile for reversing the recruitment order of peripheral nerve stimulation: a simulation study.
      can be delayed by activating slow twitch, fatigue-resistant fibers before any fast fatigable fibers are activated (Figure 2).
      • Llewellyn M.E.
      • Thompson K.R.
      • Deisseroth K.
      • Delp S.L.
      Orderly recruitment of motor units under optical control in vivo.
      • Gordon T.
      • Thomas C.K.
      • Munson J.B.
      • Stein R.B.
      The resilience of the size principle in the organization of motor unit properties in normal and reinnervated adult skeletal muscles.
      • Mendell L.M.
      The size principle: a rule describing the recruitment of motoneurons.
      Despite the substantial advantages of optical stimulation over electrical stimulation, multiple limitations must be addressed before optogenetics can be used clinically to restore function in SCI survivors.
      Figure thumbnail gr2
      Figure 2Fatigue resistance comparison between optical and electrical stimulation. A, Mean tetanic muscle tension during 2-minute stimulation with 250-ms trains of stimulation at 1 Hz using electrical and optical stimulation in 7 mice. Shaded region is standard error of the mean (average body weight [BW] = 0.258±0.01 N). B, Mean fatigue index measured as decline in tetanic muscle tension over 2 minutes in 7 mice. C, Tetanic tension from a single mouse during optical and electrical stimulation in the hind limbs over 20 minutes. ∗Indicates a statistically significant difference in muscle tension.
      Adapted by permission from Macmillan Publishers Ltd Nat Med,
      • Llewellyn M.E.
      • Thompson K.R.
      • Deisseroth K.
      • Delp S.L.
      Orderly recruitment of motor units under optical control in vivo.
      copyright 2010.

      Limitations of Optogenetic Applications

      Numerous studies using direct administration of adeno-associated virus vectors with different serotypes in small animal models have reported robust transduction rates.
      • Cederfjäll E.
      • Nilsson N.
      • Sahin G.
      • et al.
      Continuous DOPA synthesis from a single AAV: dosing and efficacy in models of Parkinson's disease.
      • Hippert C.
      • Ibanes S.
      • Serratrice N.
      • et al.
      Corneal transduction by intra-stromal injection of AAV vectors in vivo in the mouse and ex vivo in human explants.
      • Palfi A.
      • Chadderton N.
      • McKee A.G.
      • et al.
      Efficacy of codelivery of dual AAV2/5 vectors in the murine retina and hippocampus.
      • Ito T.
      • Okada T.
      • Mimuro J.
      • et al.
      Adenoassociated virus–mediated prostacyclin synthase expression prevents pulmonary arterial hypertension in rats.
      Additionally, gene therapy using viral vectors has been successfully translated to clinical practice. However, its use has uncovered multiple issues that need to be addressed before viral delivery of optogenetics can be used clinically in humans. First, efforts to reproduce efficient transduction in large animal models have been largely unsuccessful in the past. More recently, improvements in transduction efficiency have been reported in both the brain and spinal cord in swine
      • Federici T.
      • Taub J.S.
      • Baum G.R.
      • et al.
      Robust spinal motor neuron transduction following intrathecal delivery of AAV9 in pigs.
      • Passini M.A.
      • Bu J.
      • Richards A.M.
      • et al.
      Translational fidelity of intrathecal delivery of self-complementary AAV9-survival motor neuron 1 for spinal muscular atrophy.
      • Kornum B.R.
      • Stott S.R.W.
      • Mattsson B.
      • et al.
      Adeno-associated viral vector serotypes 1 and 5 targeted to the neonatal rat and pig striatum induce widespread transgene expression in the forebrain.
      and nonhuman primate models.
      • Passini M.A.
      • Bu J.
      • Richards A.M.
      • et al.
      Translational fidelity of intrathecal delivery of self-complementary AAV9-survival motor neuron 1 for spinal muscular atrophy.
      • Samaranch L.
      • Salegio E.A.
      • San Sebastian W.
      • et al.
      Strong cortical and spinal cord transduction after AAV7 and AAV9 delivery into the cerebrospinal fluid of nonhuman primates.

      Meyer K, Ferraiuolo L, Schmelzer L, et al. Improving single injection CSF delivery of AAV9-mediated gene therapy for SMA: a dose-response study in mice and nonhuman primates [published online ahead of print October 31, 2014]. Mol Ther. http://dx.doi.org/10.1038/mt.2014.210.

      Second, integration of foreign genomic material can also result in numerous adverse events including expression of proto-oncogenes
      • Vannucci L.
      • Lai M.
      • Chiuppesi F.
      • Ceccherini-Nelli L.
      • Pistello M.
      Viral vectors: a look back and ahead on gene transfer technology.
      and silencing of tumor suppressor genes,
      • High K.A.
      The gene therapy journey for hemophilia: are we there yet?.
      • Mukherjee S.
      • Thrasher A.J.
      Gene therapy for PIDs: progress, pitfalls and prospects.
      which could lead to neoplastic transformation or protein mutations leading to undesired changes in downstream cellular functions.
      • High K.A.
      The gene therapy journey for hemophilia: are we there yet?.
      • Mukherjee S.
      • Thrasher A.J.
      Gene therapy for PIDs: progress, pitfalls and prospects.
      Third, peripherally administered vectors can initiate immune responses leading to inhibition of vector function, decreased expression duration, and cytotoxic effects.
      • Mingozzi F.
      • High K.A.
      Immune responses to AAV vectors: overcoming barriers to successful gene therapy.
      • Pachter J.S.
      • de Vries H.E.
      • Fabry Z.
      The blood-brain barrier and its role in immune privilege in the central nervous system.
      • Xiao B.-G.
      • Link H.
      Immune regulation within the central nervous system.
      Current strategies to lessen immune responses include altering the capsid of the viral vector, modifying the vector delivery route, or applying techniques to inhibit or modulate immune system activity.
      • Mikals K.
      • Nam H.-J.
      • Van Vliet K.
      • et al.
      The structure of AAVrh32.33, a novel gene delivery vector.
      • Basner-Tschakarjan E.
      • Bijjiga E.
      • Martino A.T.
      Pre-clinical assessment of immune responses to adeno-associated virus (AAV) vectors.
      Alternatively, nonviral techniques could be used along with biomaterial and molecular strategies to systemically deliver genetic material into target locations.
      • Yao L.
      • Daly W.
      • Newland B.
      • et al.
      Improved axonal regeneration of transected spinal cord mediated by multichannel collagen conduits functionalized with neurotrophin-3 gene.
      • Bergen J.M.
      • Park I.-K.
      • Horner P.J.
      • Pun S.H.
      Nonviral approaches for neuronal delivery of nucleic acids.
      • Grahn P.J.
      • Vaishya S.
      • Knight A.M.
      • et al.
      Implantation of cauda equina nerve roots through a biodegradable scaffold at the conus medullaris in rat.
      Further work is also needed to identify optimal vectors (viral or nonviral) and specific administration routes for targeting specific neuronal populations. For example, efficient and selective transduction of alpha motor neurons within the ventral spinal cord will likely require intraparenchymal or intrathecal vector injection into the spinal gray matter. Alternatively, this could be achieved by retrograde transport from intraneural or intramuscular injection sites.
      Finally, multiple barriers must be overcome before chronically implantable optical systems can be developed. Some of these barriers include (1) minimizing glial responses to the implanted light guides, similar to the glial scarring observed with other long-term neural interface systems such as deep brain stimulation and intracortical recording systems; (2) optimizing light delivery paradigms to enhance temporal and spatial activation of target neurons while improving light penetration through tissue surrounding the light source
      • Vaziri A.
      • Emiliani V.
      Reshaping the optical dimension in optogenetics.
      ; and (3) reducing heating effects on tissue surrounding the light source.

      Future Directions

      Small animal studies suggest that optogenetics offers multiple advantages over electrical stimulation techniques. However, multiple steps need to be taken before optogenetics can be clinically used to restore function in SCI survivors. First, it is necessary to devise appropriate strategies for safe transgene delivery to target cell types in vivo. These strategies will require controlled transduction (via appropriate vectors and serotypes) and expression (via appropriate gene regulation promoters). Second, it is paramount to extend the expression lifetime to allow for single (or minimally repeated) administration of viral vectors and promoters. Third, stimulation systems need to be developed that optimize light delivery paradigms in a tissue-specific manner while reducing glial responses to light delivery devices. Finally, stimulation will need to be controlled in a natural manner by the user while also allowing for real-time adjustment to account for perturbations within the user’s environment.
      • Grahn P.J.
      • Mallory G.W.
      • Berry B.M.
      • Hachmann J.T.
      • Lobel D.A.
      • Lujan J.L.
      Restoration of motor function following spinal cord injury via optimal control of intraspinal microstimulation: toward a next generation closed-loop neural prosthesis.
      • Lobel D.A.
      • Lee K.H.
      Brain machine interface and limb reanimation technologies: restoring function after spinal cord injury through development of a bypass system.

      Conclusion

      Although there is still no cure for SCI, advances in stimulation and neural interfacing technology show promise for restoring neurologic function. Optogenetics offers to improve on existing FES technology by better following physiologic muscle activation, increasing selectivity, and providing simultaneous control of excitatory and inhibitory responses. In turn, advances in optogenetics technology could provide an avenue for optimal restoration of function after SCI, thereby improving the quality of life for those living with paralysis.

      Supplemental Online Material

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