Long-distance growth and connectivity of neural stem cells after severe spinal cord injury.

Neural stem cells (NSCs) expressing GFP were embedded into fibrin matrices containing growth factor cocktails and grafted to sites of severe spinal cord injury. Grafted cells differentiated into multiple cellular phenotypes, including neurons, which extended large numbers of axons over remarkable distances. Extending axons formed abundant synapses with host cells. Axonal growth was partially dependent on mammalian target of rapamycin (mTOR), but not Nogo signaling. Grafted neurons supported formation of electrophysiological relays across sites of complete spinal transection, resulting in functional recovery. Two human stem cell lines (566RSC and HUES7) embedded in growth-factor-containing fibrin exhibited similar growth, and 566RSC cells supported functional recovery. Thus, properties intrinsic to early-stage neurons can overcome the inhibitory milieu of the injured adult spinal cord to mount remarkable axonal growth, resulting in formation of new relay circuits that significantly improve function. These therapeutic properties extend across stem cell sources and species.

Emergence of functionally aberrant and subsequent reduction of neuromuscular connectivity and improved motor performance after cervical spinal cord injury in Rhesus.

Introduction: The paralysis that occurs after a spinal cord injury, particularly during the early stages of post-lesion recovery (∼6 weeks), appears to be attributable to the inability to activate motor pools well beyond their motor threshold. In the later stages of recovery, however, the inability to perform a motor task effectively can be attributed to abnormal activation patterns among motor pools, resulting in poor coordination. Method: We have tested this hypothesis on four adult male Rhesus monkeys (Macaca mulatta), ages 6-10 years, by recording the EMG activity levels and patterns of multiple proximal and distal muscles controlling the upper limb of the Rhesus when performing three tasks requiring different levels of skill before and up to 24 weeks after a lateral hemisection at C7. During the recovery period the animals were provided routine daily care, including access to a large exercise cage (5' × 7' × 10') and tested every 3-4 weeks for each of the three motor tasks. Results: At approximately 6-8 weeks the animals were able to begin to step on a treadmill, perform a spring-loaded task with the upper limb, and reaching, grasping, and eating a grape placed on a vertical stick. The predominant changes that occurred, beginning at ∼6-8 weeks of the recovery of these tasks was an elevated level of activation of most motor pools well beyond the pre-lesion level. Discussion: As the chronic phase progressed there was a slight reduction in the EMG burst amplitudes of some muscles and less incidence of co-contraction of agonists and antagonists, probably contributing to an improved ability to selectively activate motor pools in a more effective temporal pattern. Relative to pre-lesion, however, the EMG patterns even at the initial stages of recovery of successfully performing the different motor tasks, the level of activity of most muscle remained higher. Perhaps the most important concept that emerges from these data is the large combinations of adaptive strategies in the relative level of recruitment and the timing of the peak levels of activation of different motor pools can progressively provide different stages to regain a motor skill.

Rhesus macaque versus rat divergence in the corticospinal projectome.

We used viral intersectional tools to map the entire projectome of corticospinal neurons associated with fine distal forelimb control in Fischer 344 rats and rhesus macaques. In rats, we found an extraordinarily diverse set of collateral projections from corticospinal neurons to 23 different brain and spinal regions. Remarkably, the vast weighting of this "motor" projection was to sensory systems in both the brain and spinal cord, confirmed by optogenetic and transsynaptic viral intersectional tools. In contrast, rhesus macaques exhibited far heavier and narrower weighting of corticospinal outputs toward spinal and brainstem motor systems. Thus, corticospinal systems in macaques primarily constitute a final output system for fine motor control, whereas this projection in rats exerts a multi-modal integrative role that accesses far broader CNS regions. Unique structural-functional correlations can be achieved by mapping and quantifying a single neuronal system's total axonal output and its relative weighting across CNS targets.

Local and remote growth factor effects after primate spinal cord injury.

Primate models of spinal cord injury differ from rodent models in several respects, including the relative size and functional neuroanatomy of spinal projections. Fundamental differences in scale raise the possibility that retrograde injury signals, and treatments applied at the level of the spinal cord that exhibit efficacy in rodents, may fail to influence neurons at the far greater distances of primate systems. Thus, we examined both local and remote neuronal responses to neurotrophic factor-secreting cell grafts placed within sites of right C7 hemisection lesions in the rhesus macaque. Six months after gene delivery of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) into C7 lesion sites, we found both local effects of growth factors on axonal growth, and remote effects of growth factors reflected in significant reductions in axotomy-induced atrophy of large pyramidal neurons within the primary motor cortex. Additional examination in a rodent model suggested that BDNF, rather than NT-3, mediated remote protection of corticospinal neurons in the brain. Thus, injured neural systems retain the ability to respond to growth signals over the extended distances of the primate CNS, promoting local axonal growth and preventing lesion-induced neuronal degeneration at a distance. Remote cortical effects of spinally administered growth factors could "prime" the neuron to respond to experimental therapies that promote axonal plasticity or regeneration.

Quantifying the kinematic features of dexterous finger movements in nonhuman primates with markerless tracking.

Research using nonhuman primate models for human disease frequently requires behavioral observational techniques to quantify functional outcomes. The ability to assess reaching and grasping patterns is of particular interest in clinical conditions that affect the motor system (e.g., spinal cord injury, SCI). Here we explored the use of DeepLabCut, an open-source deep learning toolset, in combination with a standard behavioral task (Brinkman Board) to quantify nonhuman primate performance in precision grasping. We examined one male rhesus macaque (Macaca mulatta) in the task which involved retrieving rewards from variously-oriented shallow wells. Simultaneous recordings were made using GoPro Hero7 Black cameras (resolution 1920 x 1080 at 120 fps) from two different angles (from the side and top of the hand motion). The task/device design necessitates use of the right hand to complete the task. Two neural networks (corresponding to the top and side view cameras) were trained using 400 manually annotated images, tracking 19 unique landmarks each. Based on previous reports, this produced sufficient tracking (Side: trained pixel error of 2.15, test pixel error of 11.25; Top: trained pixel error of 2.06, test pixel error of 30.31) so that landmarks could be tracked on the remaining frames. Landmarks included in the tracking were the spatial location of the knuckles and the fingernails of each digit, and three different behavioral measures were quantified for assessment of hand movement (finger separation, middle digit extension and preshaping distance). Together, our preliminary results suggest that this markerless approach is a possible method to examine specific kinematic features of dexterous function.Clinical Relevance- The methodology presented below allows for the markerless tracking of kinematic features of dexterous finger movement by non-human primates. This method could allow for direct comparisons between human patients and non-human primate models of clinical conditions (e.g., spinal cord injury). This would provide objective quantitative metrics and crucial information for assessing movement impairments across populations and the potential translation of treatments, interventions and their outcomes.

Sexual dimorphism of detrusor function demonstrated by urodynamic studies in rhesus macaques.

The lower urinary tract (LUT) and micturition reflexes are sexually dimorphic across mammals. Sex as a biological variable is also of critical importance for the development and translation of new medical treatments and therapeutics interventions affecting pelvic organs, including the LUT. However, studies of LUT function with comparisons between the sexes have remained sparse, especially for larger mammals. Detrusor function was investigated by filling cystometry and pressure flow studies in 16 male and 22 female rhesus macaques. By filling cystometry, male subjects exhibited a significantly larger bladder capacity and compliance compared to females. Pressure flow studies showed a significantly higher bladder pressure at voiding onset, peak pressure, and elevation in detrusor-activated bladder pressure from the end of bladder filling to peak pressure in the male subjects. The activation of reflex micturition, with associated detrusor contractions, resulted in voiding in a significantly larger proportion of female compared to male subjects. A higher urethral outlet resistance is suggested in the male subjects. We conclude that sexual dimorphism of detrusor function is prominent in rhesus macaques, shares many features with the human, and merits consideration in translational and pre-clinical research studies of micturition and LUT function in non-human primates.

Regenerating Corticospinal Axons Innervate Phenotypically Appropriate Neurons within Neural Stem Cell Grafts.

Neural progenitor cell grafts form new relays across sites of spinal cord injury (SCI). Using a panel of neuronal markers, we demonstrate that spinal neural progenitor grafts to sites of rodent SCI adopt diverse spinal motor and sensory interneuronal fates, representing most neuronal subtypes of the intact spinal cord, and spontaneously segregate into domains of distinct cell clusters. Host corticospinal motor axons regenerating into neural progenitor grafts innervate appropriate pre-motor interneurons, based on trans-synaptic tracing with herpes simplex virus. A human spinal neural progenitor cell graft to a non-human primate also received topographically appropriate corticospinal axon regeneration. Thus, grafted spinal neural progenitor cells give rise to a variety of neuronal progeny that are typical of the normal spinal cord; remarkably, regenerating injured adult corticospinal motor axons spontaneously locate appropriate motor domains in the heterogeneous, developing graft environment, without a need for additional exogenous guidance.

Chondroitinase improves anatomical and functional outcomes after primate spinal cord injury.

Inhibitory extracellular matrices form around mature neurons as perineuronal nets containing chondroitin sulfate proteoglycans that limit axonal sprouting after CNS injury. The enzyme chondroitinase (Chase) degrades inhibitory chondroitin sulfate proteoglycans and improves axonal sprouting and functional recovery after spinal cord injury in rodents. We evaluated the effects of Chase in rhesus monkeys that had undergone C7 spinal cord hemisection. Four weeks after hemisection, we administered multiple intraparenchymal Chase injections below the lesion, targeting spinal cord circuits that control hand function. Hand function improved significantly in Chase-treated monkeys relative to vehicle-injected controls. Moreover, Chase significantly increased corticospinal axon growth and the number of synapses formed by corticospinal terminals in gray matter caudal to the lesion. No detrimental effects were detected. This approach appears to merit clinical translation in spinal cord injury.

Hippocampal map realignment and spatial learning.

The spatial selectivity of hippocampal neurons suggests that they contribute to an internal representation of current location. The activity of hippocampal pyramidal cells was recorded while adult (10-13 months old) and aged (24-28 months old) rats performed a task in which two spatial reference frames were put in conflict. Rats attempted to find an unmarked goal whose position was fixed relative to only one of the two reference frames. The ability of a rat's hippocampus to adjust to the conflicting information and use the 'correct' position estimate (hippocampal map 'realignment') was correlated with the rat's ability to find the hidden goal. In addition, aged rats were impaired relative to adult rats in both goal-finding accuracy and map realignment. Thus, changes in the effectiveness with which the hippocampal spatial representation is updated on the basis of external cues may contribute to both within-age-group spatial learning variability and age-related spatial learning deficits.

Activation of Intrinsic Growth State Enhances Host Axonal Regeneration into Neural Progenitor Cell Grafts.

Axonal regeneration after spinal cord injury (SCI) can be enhanced by activation of the intrinsic neuronal growth state and, separately, by placement of growth-enabling neural progenitor cell (NPC) grafts into lesion sites. Indeed, NPC grafts support regeneration of all host axonal projections innervating the normal spinal cord. However, some host axons regenerate only short distances into grafts. We examined whether activation of the growth state of the host injured neuron would elicit greater regeneration into NPC grafts. Rats received NPC grafts into SCI lesions in combination with peripheral "conditioning" lesions. Six weeks later, conditioned host sensory axons exhibited a significant, 9.6-fold increase in regeneration into the lesion/graft site compared with unconditioned axons. Regeneration was further enhanced 1.6-fold by enriching NPC grafts with phenotypically appropriate sensory neuronal targets. Thus, activation of the intrinsic host neuronal growth state and manipulation of the graft environment enhance axonal regeneration after SCI.

Restorative effects of human neural stem cell grafts on the primate spinal cord.

We grafted human spinal cord-derived neural progenitor cells (NPCs) into sites of cervical spinal cord injury in rhesus monkeys (Macaca mulatta). Under three-drug immunosuppression, grafts survived at least 9 months postinjury and expressed both neuronal and glial markers. Monkey axons regenerated into grafts and formed synapses. Hundreds of thousands of human axons extended out from grafts through monkey white matter and synapsed in distal gray matter. Grafts gradually matured over 9 months and improved forelimb function beginning several months after grafting. These findings in a 'preclinical trial' support translation of NPC graft therapy to humans with the objective of reconstituting both a neuronal and glial milieu in the site of spinal cord injury.

Delivery of neurotrophin-3 from fibrin enhances neuronal fiber sprouting after spinal cord injury.

Neurotrophins have been shown to promote axonal growth and regeneration after spinal cord injury. The therapeutic utility of neurotrophins may be enhanced by using a controlled delivery system to increase the duration of neurotrophin availability following injury. Such a delivery system can be incorporated into a bioactive scaffold to serve as a physical bridge for regeneration. This study assessed the effect of controlled delivery of neurotrophin-3 (NT-3) from fibrin scaffolds implanted in spinal cord lesions immediately following 2-mm ablation injury in adult rats. Nine days after injury, fibrin scaffolds containing the delivery system and NT-3 (1000 ng/mL) elicited more robust neuronal fiber growth into the lesion than did control scaffolds or saline (1.5- to 3-fold increase). Implantation of fibrin scaffolds resulted in a dramatic reduction of glial scar formation at the white matter border of the lesion. Hindlimb motor function of treated animals did not improve relative to controls at 12 weeks post-injury. Thus, controlled delivery of NT-3 from fibrin scaffolds enhanced the initial regenerative response by increasing neuronal fiber sprouting and cell migration into the lesion, while functional motor recovery was not observed in this model.

Impact of aging on hippocampal function: plasticity, network dynamics, and cognition.

Aging is associated with specific impairments of learning and memory, some of which are similar to those caused by hippocampal damage. Studies of the effects of aging on hippocampal anatomy, physiology, plasticity, and network dynamics may lead to a better understanding of age-related cognitive deficits. Anatomical and electrophysiological studies indicate that the hippocampus of the aged rat sustains a loss of synapses in the dentate gyrus, a loss of functional synapses in area CA1, a decrease in the NMDA-receptor-mediated response at perforant path synapses onto dentate gyrus granule cells, and an alteration of Ca(2+) regulation in area CA1. These changes may contribute to the observed age-related impairments of synaptic plasticity, which include deficits in the induction and maintenance of long-term potentiation (LTP) and lower thresholds for depotentiation and long-term depression (LTD). This shift in the balance of LTP and LTD could, in turn, impair the encoding of memories and enhance the erasure of memories, and therefore contribute to cognitive deficits experienced by many aged mammals. Altered synaptic plasticity may also change the dynamic interactions among cells in hippocampal networks, causing deficits in the storage and retrieval of information about the spatial organization of the environment. Further studies of the aged hippocampus will not only lead to treatments for age-related cognitive impairments, but may also clarify the mechanisms of learning in adult mammals.

A Unilateral Cervical Spinal Cord Contusion Injury Model in Non-Human Primates (Macaca mulatta).

The development of a non-human primate (NHP) model of spinal cord injury (SCI) based on mechanical and computational modeling is described. We scaled up from a rodent model to a larger primate model using a highly controllable, friction-free, electronically-driven actuator to generate unilateral C6-C7 spinal cord injuries. Graded contusion lesions with varying degrees of functional recovery, depending upon pre-set impact parameters, were produced in nine NHPs. Protocols and pre-operative magnetic resonance imaging (MRI) were used to optimize the predictability of outcomes by matching impact protocols to the size of each animal's spinal canal, cord, and cerebrospinal fluid space. Post-operative MRI confirmed lesion placement and provided information on lesion volume and spread for comparison with histological measures. We evaluated the relationships between impact parameters, lesion measures, and behavioral outcomes, and confirmed that these relationships were consistent with our previous studies in the rat. In addition to providing multiple univariate outcome measures, we also developed an integrated outcome metric describing the multivariate cervical SCI syndrome. Impacts at the higher ranges of peak force produced highly lateralized and enduring deficits in multiple measures of forelimb and hand function, while lower energy impacts produced early weakness followed by substantial recovery but enduring deficits in fine digital control (e.g., pincer grasp). This model provides a clinically relevant system in which to evaluate the safety and, potentially, the efficacy of candidate translational therapies.

Leveraging biomedical informatics for assessing plasticity and repair in primate spinal cord injury.

Recent preclinical advances highlight the therapeutic potential of treatments aimed at boosting regeneration and plasticity of spinal circuitry damaged by spinal cord injury (SCI). With several promising candidates being considered for translation into clinical trials, the SCI community has called for a non-human primate model as a crucial validation step to test efficacy and validity of these therapies prior to human testing. The present paper reviews the previous and ongoing efforts of the California Spinal Cord Consortium (CSCC), a multidisciplinary team of experts from 5 University of California medical and research centers, to develop this crucial translational SCI model. We focus on the growing volumes of high resolution data collected by the CSCC, and our efforts to develop a biomedical informatics framework aimed at leveraging multidimensional data to monitor plasticity and repair targeting recovery of hand and arm function. Although the main focus of many researchers is the restoration of voluntary motor control, we also describe our ongoing efforts to add assessments of sensory function, including pain, vital signs during surgery, and recovery of bladder and bowel function. By pooling our multidimensional data resources and building a unified database infrastructure for this clinically relevant translational model of SCI, we are now in a unique position to test promising therapeutic strategies' efficacy on the entire syndrome of SCI. We review analyses highlighting the intersection between motor, sensory, autonomic and pathological contributions to the overall restoration of function. This article is part of a Special Issue entitled SI: Spinal cord injury.

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.

Using templated agarose scaffolds to promote axon regeneration through sites of spinal cord injury.

The past 30 years of research in spinal cord injury (SCI) have revealed that, under certain conditions, some types of axons are able to regenerate. To aid these axons in bridging the lesion site, many experimenters place cellular grafts at the lesion. However, to increase the potential for functional recovery, it is likely advantageous to maximize the number of axons that reach the intact spinal cord on the other side of the lesion. Implanting linear-channeled scaffolds at the lesion site provides growing axons with linear growth paths, which minimizes the distance they must travel to reach healthy tissue. Moreover, the linear channels help the regenerating axons maintain the correct mediolateral and dorsoventral position in the spinal cord, which may also improve functional recovery by keeping the axons nearer to their correct targets. Here, we provide a protocol to perform a full spinal cord transection in rats that accommodates an implanted scaffold.

Neural stem cells in models of spinal cord injury.

Replication of published studies is an important and respected aspect of the conduct of science. Most would argue that the interpretation of "negative" outcomes is still more challenging than the interpretation of "positive" findings, however, due to uncertainty in knowing precisely why a hypothesized outcome was not observed: in particular, are "negative" findings in replication studies a result of invalidity of the original experimental hypothesis, or due to a methodological failure, insensitivity of the applied instruments of analysis, or other factors? These points must be carefully considered. Steward and colleagues report findings of a study in which multipotent neural progenitor cells were grafted to sites of T3 complete transection. Unlike our study, cells failed to fill the lesion site, leaving collagenous rifts between rostral and caudal graft components. This "anatomical" failure precluded formation of neural relays across the lesion site, and was predictably associated with a failure to detect functional improvement. In summarizing outcomes of the study, Steward and colleagues did not clearly link the failure to achieve graft continuity in the lesion cavity with functional outcomes, despite the central role of this observation in cogently interpreting results of the replication study. In addition, the authors stated that they failed to replicate our report of "extensive" host axonal regeneration into grafts, but we did not report "extensive" host anatomical regeneration; moreover, underexposed images may have contributed to Steward's underestimation of host axonal penetration. The authors also stated that our original study excluded some animals from functional analysis, and this is incorrect. While replication studies are important and necessary, this particular report contained several errors, and the failure to form a continuous neural progenitor cell bridge across the lesion site limited the ability to conclude whether continuous grafts can restore function. In subsequent experiments we too have observed rift formation in animals grafted at long delays (>2weeks) after SCI, and we confirm that animals with rifts do not exhibit functional improvement; we are developing methods to remove or prevent rift formation. The replication study confirmed the cardinal finding of our original report: that early-stage neural precursors extend very large numbers of axons over remarkably long distances through the lesioned adult spinal cord.

Development of a database for translational spinal cord injury research.

Efforts to understand spinal cord injury (SCI) and other complex neurotrauma disorders at the pre-clinical level have shown progress in recent years. However, successful translation of basic research into clinical practice has been slow, partly because of the large, heterogeneous data sets involved. In this sense, translational neurological research represents a "big data" problem. In an effort to expedite translation of pre-clinical knowledge into standards of patient care for SCI, we describe the development of a novel database for translational neurotrauma research known as Visualized Syndromic Information and Outcomes for Neurotrauma-SCI (VISION-SCI). We present demographics, descriptive statistics, and translational syndromic outcomes derived from our ongoing efforts to build a multi-center, multi-species pre-clinical database for SCI models. We leveraged archived surgical records, postoperative care logs, behavioral outcome measures, and histopathology from approximately 3000 mice, rats, and monkeys from pre-clinical SCI studies published between 1993 and 2013. The majority of animals in the database have measures collected for health monitoring, such as weight loss/gain, heart rate, blood pressure, postoperative monitoring of bladder function and drug/fluid administration, behavioral outcome measures of locomotion, and tissue sparing postmortem. Attempts to align these variables with currently accepted common data elements highlighted the need for more translational outcomes to be identified as clinical endpoints for therapeutic testing. Last, we use syndromic analysis to identify conserved biological mechanisms of recovery after cervical SCI between rats and monkeys that will allow for more-efficient testing of therapeutics that will need to be translated toward future clinical trials.