Recovery of walking after paralysis by regenerating characterized neurons to their natural target region.

Axon regeneration can be induced across anatomically complete spinal cord injury (SCI), but robust functional restoration has been elusive. Whether restoring neurological functions requires directed regeneration of axons from specific neuronal subpopulations to their natural target regions remains unclear. To address this question, we applied projection-specific and comparative single-nucleus RNA sequencing to identify neuronal subpopulations that restore walking after incomplete SCI. We show that chemoattracting and guiding the transected axons of these neurons to their natural target region led to substantial recovery of walking after complete SCI in mice, whereas regeneration of axons simply across the lesion had no effect. Thus, reestablishing the natural projections of characterized neurons forms an essential part of axon regeneration strategies aimed at restoring lost neurological functions.

Longitudinal interrogation of sympathetic neural circuits and hemodynamics in preclinical models.

Neurological disorders, including spinal cord injury, result in hemodynamic instability due to the disruption of supraspinal projections to the sympathetic circuits located in the spinal cord. We recently developed a preclinical model that allows the identification of the topology and dynamics through which sympathetic circuits modulate hemodynamics, supporting the development of a neuroprosthetic baroreflex that precisely controls blood pressure in rats, monkeys and humans with spinal cord injuries. Here, we describe the continuous monitoring of arterial blood pressure and sympathetic nerve activity over several months in preclinical models of chronic neurological disorders using commercially available telemetry technologies, as well as optogenetic and neuronal tract-tracing procedures specifically adapted to the sympathetic circuitry. Using a blueprint to construct a negative-pressure chamber, the approach enables the reproduction, in rats, of well-controlled and reproducible episodes of hypotension-mimicking orthostatic challenges already used in humans. Blood pressure variations can thus be directly induced and linked to the molecular, functional and anatomical properties of specific neurons in the brainstem, spinal cord and ganglia. Each procedure can be completed in under 2 h, while the construction of the negative-pressure chamber requires up to 1 week. With training, individuals with a basic understanding of cardiovascular physiology, engineering or neuroscience can collect longitudinal recordings of hemodynamics and sympathetic nerve activity over several months.

Natural and targeted circuit reorganization after spinal cord injury.

A spinal cord injury disrupts communication between the brain and the circuits in the spinal cord that regulate neurological functions. The consequences are permanent paralysis, loss of sensation and debilitating dysautonomia. However, the majority of circuits located above and below the injury remain anatomically intact, and these circuits can reorganize naturally to improve function. In addition, various neuromodulation therapies have tapped into these processes to further augment recovery. Emerging research is illuminating the requirements to reconstitute damaged circuits. Here, we summarize these natural and targeted reorganizations of circuits after a spinal cord injury. We also advocate for new concepts of reorganizing circuits informed by multi-omic single-cell atlases of recovery from injury. These atlases will uncover the molecular logic that governs the selection of 'recovery-organizing' neuronal subpopulations, and are poised to herald a new era in spinal cord medicine.

The neurons that restore walking after paralysis.

A spinal cord injury interrupts pathways from the brain and brainstem that project to the lumbar spinal cord, leading to paralysis. Here we show that spatiotemporal epidural electrical stimulation (EES) of the lumbar spinal cord1-3 applied during neurorehabilitation4,5 (EESREHAB) restored walking in nine individuals with chronic spinal cord injury. This recovery involved a reduction in neuronal activity in the lumbar spinal cord of humans during walking. We hypothesized that this unexpected reduction reflects activity-dependent selection of specific neuronal subpopulations that become essential for a patient to walk after spinal cord injury. To identify these putative neurons, we modelled the technological and therapeutic features underlying EESREHAB in mice. We applied single-nucleus RNA sequencing6-9 and spatial transcriptomics10,11 to the spinal cords of these mice to chart a spatially resolved molecular atlas of recovery from paralysis. We then employed cell type12,13 and spatial prioritization to identify the neurons involved in the recovery of walking. A single population of excitatory interneurons nested within intermediate laminae emerged. Although these neurons are not required for walking before spinal cord injury, we demonstrate that they are essential for the recovery of walking with EES following spinal cord injury. Augmenting the activity of these neurons phenocopied the recovery of walking enabled by EESREHAB, whereas ablating them prevented the recovery of walking that occurs spontaneously after moderate spinal cord injury. We thus identified a recovery-organizing neuronal subpopulation that is necessary and sufficient to regain walking after paralysis. Moreover, our methodology establishes a framework for using molecular cartography to identify the neurons that produce complex behaviours.

Epidural electrical stimulation of the cervical dorsal roots restores voluntary upper limb control in paralyzed monkeys.

Regaining arm control is a top priority for people with paralysis. Unfortunately, the complexity of the neural mechanisms underlying arm control has limited the effectiveness of neurotechnology approaches. Here, we exploited the neural function of surviving spinal circuits to restore voluntary arm and hand control in three monkeys with spinal cord injury, using spinal cord stimulation. Our neural interface leverages the functional organization of the dorsal roots to convey artificial excitation via electrical stimulation to relevant spinal segments at appropriate movement phases. Stimulation bursts targeting specific spinal segments produced sustained arm movements, enabling monkeys with arm paralysis to perform an unconstrained reach-and-grasp task. Stimulation specifically improved strength, task performances and movement quality. Electrophysiology suggested that residual descending inputs were necessary to produce coordinated movements. The efficacy and reliability of our approach hold realistic promises of clinical translation.

Preclinical upper limb neurorobotic platform to assess, rehabilitate, and develop therapies.

Numerous neurorehabilitative, neuroprosthetic, and repair interventions aim to address the consequences of upper limb impairments after neurological disorders. Although these therapies target widely different mechanisms, they share the common need for a preclinical platform that supports the development, assessment, and understanding of the therapy. Here, we introduce a neurorobotic platform for rats that meets these requirements. A four-degree-of-freedom end effector is interfaced with the rat's wrist, enabling unassisted to fully assisted execution of natural reaching and retrieval movements covering the entire body workspace. Multimodal recording capabilities permit precise quantification of upper limb movement recovery after spinal cord injury (SCI), which allowed us to uncover adaptations in corticospinal tract neuron dynamics underlying this recovery. Personalized movement assistance supported early neurorehabilitation that improved recovery after SCI. Last, the platform provided a well-controlled and practical environment to develop an implantable spinal cord neuroprosthesis that improved upper limb function after SCI.

Wireless closed-loop optogenetics across the entire dorsoventral spinal cord in mice.

Optoelectronic systems can exert precise control over targeted neurons and pathways throughout the brain in untethered animals, but similar technologies for the spinal cord are not well established. In the present study, we describe a system for ultrafast, wireless, closed-loop manipulation of targeted neurons and pathways across the entire dorsoventral spinal cord in untethered mice. We developed a soft stretchable carrier, integrating microscale light-emitting diodes (micro-LEDs), that conforms to the dura mater of the spinal cord. A coating of silicone-phosphor matrix over the micro-LEDs provides mechanical protection and light conversion for compatibility with a large library of opsins. A lightweight, head-mounted, wireless platform powers the micro-LEDs and performs low-latency, on-chip processing of sensed physiological signals to control photostimulation in a closed loop. We use the device to reveal the role of various neuronal subtypes, sensory pathways and supraspinal projections in the control of locomotion in healthy and spinal-cord injured mice.

Confronting false discoveries in single-cell differential expression.

Differential expression analysis in single-cell transcriptomics enables the dissection of cell-type-specific responses to perturbations such as disease, trauma, or experimental manipulations. While many statistical methods are available to identify differentially expressed genes, the principles that distinguish these methods and their performance remain unclear. Here, we show that the relative performance of these methods is contingent on their ability to account for variation between biological replicates. Methods that ignore this inevitable variation are biased and prone to false discoveries. Indeed, the most widely used methods can discover hundreds of differentially expressed genes in the absence of biological differences. To exemplify these principles, we exposed true and false discoveries of differentially expressed genes in the injured mouse spinal cord.

Elezanumab, a human anti-RGMa monoclonal antibody, promotes neuroprotection, neuroplasticity, and neurorecovery following a thoracic hemicompression spinal cord injury in non-human primates.

Spinal cord injury (SCI) is a devastating condition characterized by loss of function, secondary to damaged spinal neurons, disrupted axonal connections, and myelin loss. Spontaneous recovery is limited, and there are no approved pharmaceutical treatments to reduce ongoing damage or promote repair. Repulsive guidance molecule A (RGMa) is upregulated following injury to the central nervous system (CNS), where it is believed to induce neuronal apoptosis and inhibit axonal growth and remyelination. We evaluated elezanumab, a human anti-RGMa monoclonal antibody, in a novel, newly characterized non-human primate (NHP) hemicompression model of thoracic SCI. Systemic intravenous (IV) administration of elezanumab over 6 months was well tolerated and associated with significant improvements in locomotor function. Treatment of animals for 16 weeks with a continuous intrathecal infusion of elezanumab below the lesion was not efficacious. IV elezanumab improved microstructural integrity of extralesional tissue as reflected by higher fractional anisotropy and magnetization transfer ratios in treated vs. untreated animals. IV elezanumab also reduced SCI-induced increases in soluble RGMa in cerebrospinal fluid, and membrane bound RGMa rostral and caudal to the lesion. Anterograde tracing of the corticospinal tract (CST) from the contralesional motor cortex following 20 weeks of IV elezanumab revealed a significant increase in the density of CST fibers emerging from the ipsilesional CST into the medial/ventral gray matter. There was a significant sprouting of serotonergic (5-HT) fibers rostral to the injury and in the ventral horn of lower thoracic regions. These data demonstrate that 6 months of intermittent IV administration of elezanumab, beginning within 24 h after a thoracic SCI, promotes neuroprotection and neuroplasticity of key descending pathways involved in locomotion. These findings emphasize the mechanisms leading to improved recovery of neuromotor functions with elezanumab in acute SCI in NHPs.

Multi-pronged neuromodulation intervention engages the residual motor circuitry to facilitate walking in a rat model of spinal cord injury.

A spinal cord injury usually spares some components of the locomotor circuitry. Deep brain stimulation (DBS) of the midbrain locomotor region and epidural electrical stimulation of the lumbar spinal cord (EES) are being used to tap into this spared circuitry to enable locomotion in humans with spinal cord injury. While appealing, the potential synergy between DBS and EES remains unknown. Here, we report the synergistic facilitation of locomotion when DBS is combined with EES in a rat model of severe contusion spinal cord injury leading to leg paralysis. However, this synergy requires high amplitudes of DBS, which triggers forced locomotion associated with stress responses. To suppress these undesired responses, we link DBS to the intention to walk, decoded from cortical activity using a robust, rapidly calibrated unsupervised learning algorithm. This contingency amplifies the supraspinal descending command while empowering the rats into volitional walking. However, the resulting improvements may not outweigh the complex technological framework necessary to establish viable therapeutic conditions.

Neuroprosthetic baroreflex controls haemodynamics after spinal cord injury.

Spinal cord injury (SCI) induces haemodynamic instability that threatens survival1-3, impairs neurological recovery4,5, increases the risk of cardiovascular disease6,7, and reduces quality of life8,9. Haemodynamic instability in this context is due to the interruption of supraspinal efferent commands to sympathetic circuits located in the spinal cord10, which prevents the natural baroreflex from controlling these circuits to adjust peripheral vascular resistance. Epidural electrical stimulation (EES) of the spinal cord has been shown to compensate for interrupted supraspinal commands to motor circuits below the injury11, and restored walking after paralysis12. Here, we leveraged these concepts to develop EES protocols that restored haemodynamic stability after SCI. We established a preclinical model that enabled us to dissect the topology and dynamics of the sympathetic circuits, and to understand how EES can engage these circuits. We incorporated these spatial and temporal features into stimulation protocols to conceive a clinical-grade biomimetic haemodynamic regulator that operates in a closed loop. This 'neuroprosthetic baroreflex' controlled haemodynamics for extended periods of time in rodents, non-human primates and humans, after both acute and chronic SCI. We will now conduct clinical trials to turn the neuroprosthetic baroreflex into a commonly available therapy for people with SCI.

Cell type prioritization in single-cell data.

We present Augur, a method to prioritize the cell types most responsive to biological perturbations in single-cell data. Augur employs a machine-learning framework to quantify the separability of perturbed and unperturbed cells within a high-dimensional space. We validate our method on single-cell RNA sequencing, chromatin accessibility and imaging transcriptomics datasets, and show that Augur outperforms existing methods based on differential gene expression. Augur identified the neural circuits restoring locomotion in mice following spinal cord neurostimulation.

Soft Printable Electrode Coating for Neural Interfaces.

The mechanical mismatch between implantable interfaces and neural tissues may be reduced by employing soft polymeric materials. Here, we report on a simple strategy to prepare and pattern a soft electrode coating of neural interfacing devices based on a screen-printable conducting hydrogel. The coating formulation, based on polyacrylamide and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate, is suitable to additive manufacturing and exhibits excellent adhesion to polydimethylsiloxane, an elastomer commonly used as a substrate in soft neural interfaces. The soft conductive coating displays a tunable elastic modulus in the 10-100 kPa range and electrochemical properties on a par with stiff conductive inks while supporting good neural cell attachment and proliferation in vitro. Next, the soft printable hydrogel is integrated within a 4 × 4 microelectrode array for electrocorticography with 250 µm-diameter contacts. Acute recording of cortical local field potentials and electrochemical characterization preimplantation and postimplantation highlight the stability of the soft organic conductor. The overall robustness of the soft coating and its patterning method provide a promising route for a range of implantable bioelectronic applications.

Introducing a biomimetic coating for graphene neuroelectronics: towardin-vivoapplications.

Electronic micro and nano-devices are suitable tools to monitor the activity of many individual neurons over mesoscale networks. However the inorganic materials currently used in microelectronics are barely accepted by neural cells and tissues, thus limiting both the sensor lifetime and efficiency. In particular, penetrating intracortical probes face high failure rate because of a wide immune response of cells and tissues. This adverse reaction called gliosis leads to the rejection of the implanted probe after few weeks and prevent long-lasting recordings of cortical neurons. Such acceptance issue impedes the realization of many neuro-rehabilitation projects. To overcome this, graphene and related carbon-based materials have attracted a lot of interest regarding their positive impact on the adhesion and regeneration of neurons, and their ability to provide high-sensitive electronic devices, such as graphene field effect transistor (G-FET). Such devices can also be implemented on numerous suitable substrates including soft substrates to match the mechanical compliance of cells and tissues, improving further the biocompatibility of the implants. Thus, using graphene as a coating and sensing device material could significantly enhance the acceptance of intracortical probes. However, such a thin monolayer of carbon atoms could be teared off during manipulation and insertion within the brain, and could also display degradation over time. In this work, we have investigated the ability to protect graphene with a natural, biocompatible and degradable polymeric film derivated from hyaluronic acid (HA). We demonstrate that HA-based coatings can be deposited over a wide range of substrates, including intracortical probes and graphene FET arrays without altering the underlying device material, its biocompatibility and sensitivity. Moreover, we show that this coating can be monitoredin situby quantifying the number of deposited charges with the G-FET arrays. The reported graphene functionalization offers promising alternatives for improving the acceptance of various neural interfaces.

Monolayer Graphene Coating of Intracortical Probes for Long-Lasting Neural Activity Monitoring.

The invasiveness of intracortical interfaces currently used today is responsible for the formation of an intense immunoresponse and inflammatory reaction from neural cells and tissues. This leads to a high concentration of reactive glial cells around the implant site, creating a physical barrier between the neurons and the recording channels. Such a rejection of foreign analog interfaces causes neural signals to fade from recordings which become flooded by background noise after a few weeks. Despite their invasiveness, those devices are required to track single neuron activity and decode fine sensory or motor commands. In particular, such quantitative and long-lasting recordings of individual neurons are crucial during a long time period (several months) to restore essential functions of the cortex, disrupted after injuries, stroke, or neurodegenerative diseases. To overcome this limitation, graphene and related materials have attracted numerous interests, as they gather in the same material many suitable properties for interfacing living matter, such as an exceptionally high neural affinity, diffusion barrier, and high physical robustness. In this work, the neural affinity of a graphene monolayer with numerous materials commonly used in neuroprostheses is compared, and its impact on the performance and durability of intracortical probes is investigated. For that purpose, an innovative coating method to wrap 3D intracortical probes with a continuous monolayer graphene is developed. Experimental evidence demonstrate the positive impact of graphene on the bioacceptance of conventional intracortical probes, in terms of detection efficiency and tissues responses, allowing real-time samplings of motor neuron activity during 5 weeks. Since continuous graphene coatings can easily be implemented on a wide range of 3D surfaces, this study further motivates the use of graphene and related materials as it could significantly contribute to reduce the current rejection of neural probes currently used in many research areas, from fundamental neurosciences to medicine and neuroprostheses.

Cbp-dependent histone acetylation mediates axon regeneration induced by environmental enrichment in rodent spinal cord injury models.

After a spinal cord injury, axons fail to regenerate in the adult mammalian central nervous system, leading to permanent deficits in sensory and motor functions. Increasing neuronal activity after an injury using electrical stimulation or rehabilitation can enhance neuronal plasticity and result in some degree of recovery; however, the underlying mechanisms remain poorly understood. We found that placing mice in an enriched environment before an injury enhanced the activity of proprioceptive dorsal root ganglion neurons, leading to a lasting increase in their regenerative potential. This effect was dependent on Creb-binding protein (Cbp)-mediated histone acetylation, which increased the expression of genes associated with the regenerative program. Intraperitoneal delivery of a small-molecule activator of Cbp at clinically relevant times promoted regeneration and sprouting of sensory and motor axons, as well as recovery of sensory and motor functions in both the mouse and rat model of spinal cord injury. Our findings showed that the increased regenerative capacity induced by enhancing neuronal activity is mediated by epigenetic reprogramming in rodent models of spinal cord injury. Understanding the mechanisms underlying activity-dependent neuronal plasticity led to the identification of potential molecular targets for improving recovery after spinal cord injury.

Cortico-reticulo-spinal circuit reorganization enables functional recovery after severe spinal cord contusion.

Severe spinal cord contusions interrupt nearly all brain projections to lumbar circuits producing leg movement. Failure of these projections to reorganize leads to permanent paralysis. Here we modeled these injuries in rodents. A severe contusion abolished all motor cortex projections below injury. However, the motor cortex immediately regained adaptive control over the paralyzed legs during electrochemical neuromodulation of lumbar circuits. Glutamatergic reticulospinal neurons with residual projections below the injury relayed the cortical command downstream. Gravity-assisted rehabilitation enabled by the neuromodulation therapy reinforced these reticulospinal projections, rerouting cortical information through this pathway. This circuit reorganization mediated a motor cortex-dependent recovery of natural walking and swimming without requiring neuromodulation. Cortico-reticulo-spinal circuit reorganization may also improve recovery in humans.

A brain-spine interface alleviating gait deficits after spinal cord injury in primates.

Spinal cord injury disrupts the communication between the brain and the spinal circuits that orchestrate movement. To bypass the lesion, brain-computer interfaces have directly linked cortical activity to electrical stimulation of muscles, and have thus restored grasping abilities after hand paralysis. Theoretically, this strategy could also restore control over leg muscle activity for walking. However, replicating the complex sequence of individual muscle activation patterns underlying natural and adaptive locomotor movements poses formidable conceptual and technological challenges. Recently, it was shown in rats that epidural electrical stimulation of the lumbar spinal cord can reproduce the natural activation of synergistic muscle groups producing locomotion. Here we interface leg motor cortex activity with epidural electrical stimulation protocols to establish a brain-spine interface that alleviated gait deficits after a spinal cord injury in non-human primates. Rhesus monkeys (Macaca mulatta) were implanted with an intracortical microelectrode array in the leg area of the motor cortex and with a spinal cord stimulation system composed of a spatially selective epidural implant and a pulse generator with real-time triggering capabilities. We designed and implemented wireless control systems that linked online neural decoding of extension and flexion motor states with stimulation protocols promoting these movements. These systems allowed the monkeys to behave freely without any restrictions or constraining tethered electronics. After validation of the brain-spine interface in intact (uninjured) monkeys, we performed a unilateral corticospinal tract lesion at the thoracic level. As early as six days post-injury and without prior training of the monkeys, the brain-spine interface restored weight-bearing locomotion of the paralysed leg on a treadmill and overground. The implantable components integrated in the brain-spine interface have all been approved for investigational applications in similar human research, suggesting a practical translational pathway for proof-of-concept studies in people with spinal cord injury.

Spatiotemporal neuromodulation therapies engaging muscle synergies improve motor control after spinal cord injury.

Electrical neuromodulation of lumbar segments improves motor control after spinal cord injury in animal models and humans. However, the physiological principles underlying the effect of this intervention remain poorly understood, which has limited the therapeutic approach to continuous stimulation applied to restricted spinal cord locations. Here we developed stimulation protocols that reproduce the natural dynamics of motoneuron activation during locomotion. For this, we computed the spatiotemporal activation pattern of muscle synergies during locomotion in healthy rats. Computer simulations identified optimal electrode locations to target each synergy through the recruitment of proprioceptive feedback circuits. This framework steered the design of spatially selective spinal implants and real-time control software that modulate extensor and flexor synergies with precise temporal resolution. Spatiotemporal neuromodulation therapies improved gait quality, weight-bearing capacity, endurance and skilled locomotion in several rodent models of spinal cord injury. These new concepts are directly translatable to strategies to improve motor control in humans.

Pronounced species divergence in corticospinal tract reorganization and functional recovery after lateralized spinal cord injury favors primates.

Experimental and clinical studies suggest that primate species exhibit greater recovery after lateralized compared to symmetrical spinal cord injuries. Although this observation has major implications for designing clinical trials and translational therapies, advantages in recovery of nonhuman primates over other species have not been shown statistically to date, nor have the associated repair mechanisms been identified. We monitored recovery in more than 400 quadriplegic patients and found that functional gains increased with the laterality of spinal cord damage. Electrophysiological analyses suggested that corticospinal tract reorganization contributes to the greater recovery after lateralized compared with symmetrical injuries. To investigate underlying mechanisms, we modeled lateralized injuries in rats and monkeys using a lateral hemisection, and compared anatomical and functional outcomes with patients who suffered similar lesions. Standardized assessments revealed that monkeys and humans showed greater recovery of locomotion and hand function than did rats. Recovery correlated with the formation of corticospinal detour circuits below the injury, which were extensive in monkeys but nearly absent in rats. Our results uncover pronounced interspecies differences in the nature and extent of spinal cord repair mechanisms, likely resulting from fundamental differences in the anatomical and functional characteristics of the motor systems in primates versus rodents. Although rodents remain essential for advancing regenerative therapies, the unique response of the primate corticospinal tract after injury reemphasizes the importance of primate models for designing clinically relevant treatments.