Human Spinal Cord Research Articles

Topographic and Anatomical Features of the Human Vertebral Column and Spinal Cord at the 18–21 Weeks of Fetal Ontogenesis

The aim of study was to identify anatomical features of the vertebral column and spinal cord, as well as their relationships at the stage of the intermediate fetal period of ontogenesis.Material and methods. The research included the study and analysis of the sectional material of 20 human fetuses of both genders at the 18–21 weeks of their development. The material was obtained as a result of abortion on social indications. The complex of morphological methods such as macro-microscopic preparation, N.I. Pirogov technique of preparations of variously-planared sections, preparation of series of histotopogrammes with van Gieson’s staining was used in the study.Results. The study has revealed that the length of the vertebral column varied from 110±4.9 mm to 122±4.7 mm in the investigated period of ontogenesis. The development of the vertebral column curvatures was observed; thoracic kyphosis being found most commonly (in 80% of cases). The transverse size of the vertebral body was larger than the anterior-posterior size of the vertebral column. There was an asymmetry of holes in the transverse processes of the cervical vertebrae. The character of changes in the vertebral canal was defined, the dynamics of these changes was quantitatively described. The morphometry of the spinal cord was performed in correlation with the spinal column. The largest length of the spinal roots was registered at the level of Sc5. The study demonstrated that development of the spinal cord is subject to the rule of bilateral asymmetry: spinal roots of the same segment on the right and left sides are not always symmetrical and may have different length parameters.Conclusion. Thus, the obtained data on quantitative morphometry and topography of the vertebral column and spinal cord at the 18–21 weeks of the human ontogenesis can be used as the anatomical basis for the developing fetal surgery and diagnostic methods.

Spatiotemporal gene expression in ALS

Human Genomics Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease that affects nerve cells in the brain and the spinal cord. It has proven difficult to identify the early stages of disease and where it spreads within the body. Maniatis et al. used RNA sequencing to define transcriptomic changes over the course of disease in different regions of the spinal cord of a mouse ALS model and a postmortem human ALS spinal cord. From changes in gene expression, they identified disease-associated pathways and established the key steps in motor neuron degeneration observed in ALS. Science , this issue p. [89][1] [1]: /lookup/volpage/364/89?iss=6435

Spatiotemporal dynamics of molecular pathology in amyotrophic lateral sclerosis.

Paralysis occurring in amyotrophic lateral sclerosis (ALS) results from denervation of skeletal muscle as a consequence of motor neuron degeneration. Interactions between motor neurons and glia contribute to motor neuron loss, but the spatiotemporal ordering of molecular events that drive these processes in intact spinal tissue remains poorly understood. Here, we use spatial transcriptomics to obtain gene expression measurements of mouse spinal cords over the course of disease, as well as of postmortem tissue from ALS patients, to characterize the underlying molecular mechanisms in ALS. We identify pathway dynamics, distinguish regional differences between microglia and astrocyte populations at early time points, and discern perturbations in several transcriptional pathways shared between murine models of ALS and human postmortem spinal cords.

The Effect of Non-Gabapentinoid Anticonvulsants on Sensorimotor Recovery After Human Spinal Cord Injury.

Recent observational studies have shown an association between gabapentinoid anticonvulsants and greater motor recovery after spinal cord injury. There is preclinical evidence to suggest that other anticonvulsants, such as sodium channel blockers, may also confer beneficial effects. The aim of the current study was to determine if non-gabapentinoid anticonvulsants were associated with neurological recovery after acute, traumatic spinal cord injury. This was an observational cohort study using data from the Sygen clinical trial. The primary outcome was total motor score recovery in the first year after injury. Anticonvulsant use was extracted from concomitant medication records; individuals were classified based on early administration (within 30days of injury), or late/no administration. Motor recovery was compared using linear mixed effects regression models with a drug-by-time interaction, and adjustment for confounders. A secondary analysis incorporated a propensity score matched cohort. Of the cohort (n = 570), 6% received anticonvulsants (carbamazepine, phenytoin, clonazepam, phenobarbital, and valproic acid) early after injury. After adjustments for initial injury level and severity, early exposure to non-gabapentinoid anticonvulsants was not associated with motor neurological outcomes (p = 0.38 for all anticonvulsants, p = 0.83 for sodium channel blockers, p = 0.82 in propensity-matched cohort). Non-gabapentinoid anticonvulsant exposure was not associated with greater or lesser neurological recovery. This suggests that these medications, as administered for the acute management of spinal cord injury, do not impact long-term neurological outcomes.

Multicenter, Randomized, Double-Blind Study Protocol Using Human Spinal Cord Recording Comparing Safety, Efficacy, and Neurophysiological Responses Between Patients Being Treated With Evoked Compound Action Potential–Controlled Closed-Loop Spinal Cord Stimulation or Open-Loop Spinal Cord Stimulation (the Evoke Study)

The spinal cord (SC) response to stimulation has yet to be studied in a pivotal clinical study. We report the study design of an ongoing multicenter, randomized, double-blind, controlled, parallel-arm study of an evoked compound action potential (ECAP) controlled closed-loop spinal cord stimulation (SCS) system, which aims to gain U.S. Food and Drug Administration approval. This study will enroll 134 SCS candidates with chronic trunk and limb pain from up to 20 United States sites. Subjects are randomized 1:1 to receive ECAP-controlled closed-loop or open-loop, conventional SCS. The primary objective is noninferiority of closed-loop stimulation determined by the proportion of subjects with ≥50% reduction in overall trunk and limb pain and no increase in pain medications at the three-month visit. If noninferiority is met, superiority is tested. In addition, measures recommended by IMMPACT (e.g., pain intensity, functional disability, emotional functioning, quality of life, impression of change, and sleep), neurophysiological properties (e.g., SC activation, conduction velocity, chronaxie, and rheobase), and safety are analyzed. All approved SCS therapies, regardless of the presence or absence of stimulation induced paresthesias, produce fixed-output stimuli; that is, the energy delivered from the electrode array has a defined output irrespective of the neural response of SC fibers. An SCS system has been developed that directly measures the neurophysiologic activation of the SC to stimulation (i.e., ECAP amplitude) and uses this information in a feedback mechanism to produce closed-loop SCS to maintain optimal and stable activation of the SC. This study represents the first randomized, double-blind, pivotal study in the field of neuromodulation to measure SC activation in ECAP-controlled closed-loop versus open-loop stimulation and is expected to yield important information regarding differences in safety, efficacy, and neurophysiological properties. The potential clinical utility of these objective measurements of SC activation and other neurophysiological properties promises to improve outcomes of SCS for chronic pain patients.

Measurement of T2* in the human spinal cord at 3T.

To measure the transverse relaxation time T2* in healthy human cervical spinal cord gray matter (GM) and white matter (WM) at 3T. Thirty healthy volunteers were recruited. Axial images were acquired using an averaged multi-echo gradient-echo (mFFE) T2*-weighted sequence with 5 echoes. We used the signal equation for an mFFE sequence with constant dephasing gradients after each echo to jointly estimate the spin density and T2* for each voxel. No global difference in T2* was observed between all GM (41.3 ± 5.6ms) and all WM (39.8 ± 5.4ms). No significant differences were observed between left (43.2 ± 6.8ms) and right (43.4 ± 5.5ms) ventral GM, left (38.3 ± 6.1ms) and right (38.6 ± 6.5ms) dorsal GM, and left (39.4 ± 5.8ms) and right (40.3 ± 5.8ms) lateral WM. However, significant regional differences were observed between ventral (43.4 ± 5.7ms) and dorsal (38.4 ± 6.0ms) GM (p<0.05), as well as between ventral (42.9 ± 6.5ms) and dorsal (37.9 ± 6.2ms) WM (p<0.05). In analyses across slices, inferior T2* was longer than superior T2* in GM (44.7ms vs. 40.1ms; p<0.01) and in WM (41.8ms vs. 35.9ms; p<0.01). Significant differences in T2* are observed between ventral and dorsal GM, ventral and dorsal WM, and superior and inferior GM and WM. There is no evidence for bilateral asymmetry in T2* in the healthy cord. These values of T2* in the spinal cord are notably lower than most reported values of T2* in the cortex.

Wnt Signaling Alterations in the Human Spinal Cord of Amyotrophic Lateral Sclerosis Cases: Spotlight on Fz2 and Wnt5a.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with no cure, and elucidation of the mechanisms mediating neuronal death in this neuropathology is crucial to develop effective treatments. It has recently been demonstrated in animal models that the Wnt family of proteins is involved in this neuropathology, although its potential involvement in case of humans is almost unknown. We analyzed the expression of Wnt signaling components in healthy and ALS human spinal cords by quantitative RT-PCR, and we found that most Wnt ligands, modulators, receptors, and co-receptors were expressed in healthy controls. Moreover, we observed clear alterations in the mRNA expression of different components of this family of proteins in human spinal cord tissue from ALS cases. Specifically, we detected a significant increase in the mRNA levels of Wnt3, Wnt4, Fz2, and Fz8, together with several non-significant increases in the mRNA expression of other genes such as Wnt2b, Wnt5a, Fz3, Lrp5, and sFRP3. Based on these observations and on previous reports of studies performed in animal models, we evaluated with immunohistochemistry the protein expression patterns of Fz2 and Fz5 receptors and their main ligand Wnt5a in control samples and ALS cases. No substantial changes were observed in Fz5 protein expression pattern in ALS samples. However, we detected an increase in the amount of Fz2+ astrocytes in the borderline between gray and white matter at the ventral horn in ALS samples. Finally, Wnt5a expression was observed in neurons and astrocytes in both control and ALS samples, although Wnt5a immunolabeling in astroglial cells was significantly increased in ALS spinal cords in the same region where changes in Fz2 were observed. Altogether, these observations strongly suggest that the Wnt family of proteins, and more specifically Fz2 and Wnt5a, might be involved in human ALS pathology.

The Human Spinal Cord is a Promising Target for Allogeneic Neural Stem Cell Transplantation

This report documents the first-in-man investigational trial and long-term follow-up of HuCNS-SC transplantation in chronic SCI. The small study size and open-label design reflect the pilot nature of this investigation, and the focus on determining feasibility, safety and preliminary efficacy. Based on the short- and long-term safety profile established in this study for individuals with thoracic SCI, a larger-scale, randomized, study in people with cervical SCI was approved in the US. These trials have established that the human spinal cord is a promising target for neural stem cell transplantation.

Coordinated Networks in the Human Brainstem and Spinal Cord during the Expectation of Pain

POSTER ABSTRACTIntroduction/Aim: Spontaneous variations in activity of brainstem (BS) and spinal cord (SC) regions may arise from a number of functions such as autonomic regulation, sensory, and motor functions. Recent evidence suggests that changes in a person’s cognitive/emotional state are linked to changes in identified BS and SC resting-state networks, indicating that these networks likely play a role in the integration of homeostatic autonomic functions. The aim of this study was to investigate how these networks change when participants are specifically expecting pain.Methods: Previously, data were obtained from the cervical SC and brainstem in 17 healthy participants during a stimulation paradigm that involved a predictable noxious heat stimulus. Blood oxygenation-level dependent (BOLD) fMRI data were obtained at 3 Tesla, with T2-weighted single-shot fast spin-echo imaging. For the current study we investigated functional connectivity in the entire 3D region with structural equation modelling (SEM) during the first two minutes of each run (baseline period, and after participants were told whether to expect a painful stimulus).Results: SEM results showed extensive connectivity within and across BS and SC regions both when participants were expecting pain, and when they were expecting no pain. Furthermore, significant differences in connectivity between regions of the BS and SC were also identified across study conditions.Discussion/Conclusions: The results indicate that connectivity across BS/SC networks is influenced by the expectation of pain in specific ways. The known functions of the regions involved support the conclusion that these networks likely serve to integrate autonomic regulation functions with pain processing.

Development and characterization of sphingosine 1-phosphate receptor 1 monoclonal antibody suitable for cell imaging and biochemical studies of endogenous receptors.

Although sphingosine-1-phosphate receptor 1 (S1P1) has been shown to trigger several S1P targeted functions such as immune cell trafficking, cell proliferation, migration, or angiogenesis, tools that allow the accurate detection of endogenous S1P1 localization and trafficking remain to be obtained and validated. In this study, we developed and characterized a novel monoclonal S1P1 antibody. Mice were immunized with S1P1 produced in the yeast Pichia pastoris and nine hybridoma clones producing monoclonal antibodies were created. Using different technical approaches including Western blot, immunoprecipitation and immunocytochemistry, we show that a selected clone, hereinafter referred to as 2B9, recognizes human and mouse S1P1 in various cell lineages. The interaction between 2B9 and S1P1 is specific over receptor subtypes, as the antibody does not binds to S1P2 or S1P5 receptors. Using cell-imaging methods, we demonstrate that 2B9 binds to an epitope located at the intracellular domain of S1P1; reveals cytosolic and membrane localization of the endogenous S1P1; and receptor internalization upon S1P or FTY720-P stimulation. Finally, loss of 2B9 signal upon knockdown of endogenous S1P1 by specific small interference RNAs further confirms its specificity. 2B9 was also able to detect S1P1 in human kidney and spinal cord tissue by immunohistochemistry. Altogether, our results suggest that 2B9 could be a useful tool to detect, quantify or localize low amounts of endogenous S1P1 in various physiological and pathological processes.

Transspinal stimulation increases motoneuron output of multiple segments in human spinal cord injury.

Targeted neuromodulation strategies that strengthen neuronal activity are in great need for restoring sensorimotor function after chronic spinal cord injury (SCI). In this study, we established changes in the motoneuron output of individuals with and without SCI after repeated noninvasive transspinal stimulation at rest over the thoracolumbar enlargement, the spinal location of leg motor circuits. Cases of motor incomplete and complete SCI were included to delineate potential differences when corticospinal motor drive is minimal. All 10 SCI and 10 healthy control subjects received daily monophasic transspinal stimuli of 1-ms duration at 0.2 Hz at right soleus transspinal evoked potential (TEP) subthreshold and suprathreshold intensities at rest. Before and two days after cessation of transspinal stimulation, we determined changes in TEP recruitment input-output curves, TEP amplitude at stimulation frequencies of 0.1, 0.125, 0.2, 0.33 and 1.0 Hz, and TEP postactivation depression upon transspinal paired stimuli at interstimulus intervals of 60, 100, 300, and 500 ms. TEPs were recorded at rest from bilateral ankle and knee flexor/extensor muscles. Repeated transspinal stimulation increased the motoneuron output over multiple segments. In control and complete SCI subjects, motoneuron output increased for knee muscles, while in motor incomplete SCI subjects motoneuron output increased for both ankle and knee muscles. In control subjects, TEPs homosynaptic and postactivation depression were present at baseline, and were potentiated for the distal ankle or knee flexor muscles. TEPs homosynaptic and postactivation depression at baseline depended on the completeness of the SCI, with minimal changes observed after transspinal stimulation. These results indicate that repeated transspinal stimulation increases spinal motoneuron responsiveness of ankle and knee muscles in the injured human spinal cord, and thus can promote motor recovery. This noninvasive neuromodulation method is a promising modality for promoting functional neuroplasticity after SCI.

CNS-derived extracellular vesicles from superoxide dismutase 1 (SOD1)G93A ALS mice originate from astrocytes and neurons and carry misfolded SOD1

Extracellular vesicles (EVs) are secreted by myriad cells in culture and also by unicellular organisms, and their identification in mammalian fluids suggests that EV release also occurs at the organism level. However, although it is clearly important to better understand EVs' roles in organismal biology, EVs in solid tissues have received little attention. Here, we modified a protocol for EV isolation from primary neural cell culture to collect EVs from frozen whole murine and human neural tissues by serial centrifugation and purification on a sucrose gradient. Quantitative proteomics comparing brain-derived EVs from nontransgenic (NTg) and a transgenic amyotrophic lateral sclerosis (ALS) mouse model, superoxide dismutase 1 (SOD1)G93A, revealed that these EVs contain canonical exosomal markers and are enriched in synaptic and RNA-binding proteins. The compiled brain EV proteome contained numerous proteins implicated in ALS, and EVs from SOD1G93A mice were significantly depleted in myelin-oligodendrocyte glycoprotein compared with those from NTg animals. We observed that brain- and spinal cord-derived EVs, from NTg and SOD1G93A mice, are positive for the astrocyte marker GLAST and the synaptic marker SNAP25, whereas CD11b, a microglial marker, was largely absent. EVs from brains and spinal cords of the SOD1G93A ALS mouse model, as well as from human SOD1 familial ALS patient spinal cord, contained abundant misfolded and nonnative disulfide-cross-linked aggregated SOD1. Our results indicate that CNS-derived EVs from an ALS animal model contain pathogenic disease-causing proteins and suggest that brain astrocytes and neurons, but not microglia, are the main EV source.

A Study to Find Normal Dimensions of Spinal Cord on MRI in Indian Population

Background: Knowledge of normal measurements of the spinal cord is essential in diagnosing and interpreting various spinal disorders. There is sparse data on normal morphometry of spinal cord in Indian population. Aims and objectives: To obtain dimensions of the human spinal cord, at various vertebral levels&nbsp; in normal population on MRI. Subjects and Methods: A cross sectional observational study was done on 60 healthy subjects aged between 20 to 40 years who underwent MRI in a tertiary care centre from December 2017 to Dec 2018. Dimensions of the multiple segments of human spinal cord were measured in Antero-posterior and transverse diameters at each level by high resolution T2-weighted images by 1.5 T Philips MR System Achieva. Results: Mean age of subjects was 28 years. In the cervical segment, the AP diameter of the spinal cord was greatest at C1 (7.74 mm in males, 6.63 mm in females) and lowest at C7. The Transverse diameter decreased from C1 to C2 level, and then increased from C2 to C5, with C5 (12.84 mm in males and 11.55 mm in females) being the maximum enlarged segment and then decreased towards C7. In the upper thoracic cord, the AP diameter and transverse diameter was maximum at D1 and decreased gradually from D1 to D6. In the lower thoracic cord, the AP diameter and transverse diameter was maximum at D12. Conclusion: Spinal cord dimensions vary at different segments and also between males and females. Hence, there is a need to establish normal reference values of spinal cord diameters at various levels.

Synergistic influences of sensory and central stimuli on non-voluntary rhythmic arm movements

In recent years, neuromodulation of the cervical spinal circuitry has become an area of interest for investigating rhythmogenesis of the human spinal cord and interaction between cervical and lumbosacral circuitries, given the involvement of rhythmic arm muscle activity in many locomotor tasks. We have previously shown that arm muscle vibrostimulation can elicit non-voluntary upper limb oscillations in unloading body conditions. Here we investigated the excitability of the cervical spinal circuitry by applying different peripheral and central stimuli in healthy humans. The rationale for applying combined stimuli is that the efficiency of only one stimulus is generally limited. We found that low-intensity electrical stimulation of the superficial arm median nerve can evoke rhythmic arm movements. Furthermore, the movements were enhanced by additional peripheral stimuli (e.g., arm muscle vibration, head turns or passive rhythmic leg movements). Finally, low-frequency transcranial magnetic stimulation of the motor cortex significantly facilitated rhythmogenesis. The findings are discussed in the general framework of a brain-spinal interface for developing adaptive central pattern generator-modulating therapies.

Safety and Preliminary Efficacy of Allogeneic Neural Stem Cell Transplantation in Chronic Spinal Cord Injury: A Translational Phase I/IIa Trial

Background: Given the large number of individuals living with disabling sensorimotor impairment due to spinal cord injury (SCI) approaching 2 million, viable options for regenerative repair strategies of the injured spinal cord are desperately needed. Human central nervous system stem cells (HuCNS-SC®) are self-renewing, multi-potent adult stem cells exhibiting successful engraftment, migration, and region-appropriate differentiation as demonstrated in multiple animal models for the injured brain and spinal cord. In preclinical SCI models, improved locomotor function provided the rationale for the first human thoracic SCI clinical trial of HuCNS-SC cells. Methods: Safety and preliminary efficacy data were collected within a first ever multi-site phase I/IIa trial involving surgical transplantation of 20 million HuCNS-SC cells into the thoracic cord in 12 subjects with sensory or motor complete traumatic paraplegia. Up to 6 years follow-up data was collected applying an extended protocol design augmenting clinical assessments through corroborating measures of sensory thresholds and neuroimaging to improve readouts of safety and efficacy. Findings: The study revealed promising short and long term surgical and medical safety. Measures of preliminary efficacy could discern responders (5/12 patients) with reliable improvements in sensory function albeit without measurable motor changes in thoracic injuries. Imaging of the spinal cord at and rostral the site of injection revealed dynamic structural changes similar to findings in non-transplanted SCI subjects. Interpretation: Findings of safety and feasibility supported the regulatory approval for a Phase II HuCNS-SC transplantation study in cervical spinal cord injured patients. Funding: The initial study (short term follow-up of the first year; STFU) and part of the second study (long term follow-up; LTFU) was funded by Stem Cells Inc, Newark, California, USA. After dissolution of Stem Cells Inc. Q3 2016 grants from Wings for Life (WfL), and the Bright Oceans Corporation (BOCO) enabled completion of the LTFU, compilation of the full set of monitored data (initial Phase I/IIa STFU trial and LTFU study), data analysis and manuscript completion. Declaration of Interest: Armin Curt has no competing interests. Principal Investigator for multi-site Phase I/IIa trial of 12 subjects and primary author (corresponding author). Jane Hsieh, with no competing interests, is an independent consultant to the Spinal Cord Injury Center Balgrist initiating data analysis and the first draft of the manuscript, as well as coordinating contributions from collaborating authors. Martin Schubert with no competing interests, participated in care of 9 enrolled subjects, data analysis and review of manuscript. Markus Hupp has no competing interests. Participated in the care of 9 enrolled subjects and reviewed the manuscript. Susi Friedl has no competing interests. Participated in the care of 9 enrolled subjects and reviewed the manuscript. Patrick Freund has no competing interests. Collected and analyzed neuroimaging data for 9 enrolled subjects and reviewed the manuscript. Eveline Huber has no competing interests. Collected and analyzed neuroimaging data for 9 enrolled subjects and reviewed the manuscript. Dario Pfyffer has no competing interests. Collected and analyzed neuroimaging data for 9 enrolled subjects, assisted with figure creation and reviewed the manuscript. Safety of neural stem cell transplantation into the human spinal cord Draft version: V6.1 20190128 Reto Sutter has no competing interests. Collected and analysed neuroimaging data for 9 enrolled subjects and reviewed the manuscript. Catherine Jutzeler has no competing interests. Collected and analysed electrophysiological data for 9 enrolled subjects and reviewed the manuscript. Rudolf P. Wuthrich has no competing interests. Provided immunosuppression for 9 enrolled subjects and reviewed the manuscript. Kan Min has no competing conflicts of interest. Performed orthopaedic spinal surgery for 9 HuCNS-SC transplantations at Balgrist site and reviewed the manuscript. Steve Casha has no competing interests. Site investigator at University of Calgary with 2 patients enrolled and transplanted, and reviewed the manuscript. Michael Fehlings has no competing interests. Site investigator at University of Toronto with 1 patient enrolled and transplanted, and reviewed the manuscript. Raphael Guzmann has no competing interests. Performed neurosurgery and HuCNS-SC transplantation for 9 enrolled subjects and reviewed the manuscript. Ethical Approval: The study protocol and all amendments were approved by related Ethics Committees and regulatory agencies in accordance with the Declaration of Helsinki.

Investigation of Resting-State BOLD Networks in the Human Brainstem and Spinal Cord

Resting-state functional magnetic resonance imaging (rs-fMRI) has been used to investigate networks within the cortex and has also provided some insight into the networks present in the brainstem (BS) and spinal cord (SC). The purpose of this study was to investigate resting-state blood oxygenation-level dependent (BOLD) fluctuations in the BS/SC and to identify resting-state networks (RSNs) across these regions. Resting-state BOLD fMRI data were obtained from the entire BS and cervical SC in 16 healthy participants, at 3 T, with T2-weighted single-shot fast spin-echo imaging. Data were spatially normalized and processed to remove physiological noise. Cluster-cluster functional connectivity was investigated across the entire 3D region by means of temporal correlations, and structural equation modeling (SEM) was used to investigate RSNs. Extensive connectivity was observed within and across BS and SC regions, with connections spanning up to 120 mm, although shorter connections were more prevalent. SEM results revealed extensive brainstem-cord connectivity that included specific anatomical regions within the brainstem. The results indicate the presence of a complex resting-state network which is highly interconnected in the spinal cord. Known anatomical connections between cortical and BS regions support the conclusion that the observed resting-state BOLD fluctuations in the BS/SC may be related to autonomic regulation. Future studies are required to further investigate these resting-state BOLD networks.

Time course and expression pattern of the neuronal markers in the developing human spinal cord

The aim of this study was to examine the spatio-temporal appearance of different neuronal cell subtypes by analyzing expression patterns of several neuronal markers (calretinin, neurofilament 200 (NF200), vanilloid receptor 1(VR1) and calcitonin gene-related peptide (CGRP)) of the embryonic human spinal cord (SC). Developing human SCs from 11 human conceptuses beetwen 5–10 developmental weeks (DW) were examined by light and electron microscopy and immunofluorescence. Light and electron microscopy revealed different embryonic stages of recognizable structure of the SC. NF200, CGRP and VR1 positive cells were observed in SCs during 5th–6th DW. NF200 was predominantly expressed in the ventral part, indicating presence of motoneurons. As development advanced, NF200 was mainly expressed in the marginal zone. Expression of CGRP was intense during all of the investigated periods, predominantly during the 5th–6th DW pointing to neural sensory differentiation, as opposed to the last DW when reduced expression of CGRP in the marginal layer indicated the terminations of the sensory afferents. Expression of VR1 was highest in the intermediate zone, at the beginning and at the end of the investigated periods, pointing to VR1 spatial pattern in the visceral afferents in the grey matter, while the first signs of calretinin were found in the 9th–10th DW ventrally. Delineating the relationships between factors involved in processes of neuronal differentiation as well as spatial and temporal arrangement of SC interrelated neurons can provide a useful information about normal SC development as well as the insight in possible causes of anomalies and disorders during embryonic life.

Generation 2 kilohertz spinal cord stimulation (kHz-SCS) bioheat multi-physics model

Kilohertz frequency spinal cord stimulation (kHz-SCS) deposits significantly more power in tissue compared to SCS at conventional frequencies, reflecting increased duty cycle (pulse compression). We hypothesize that kHz-SCS increases local tissue temperature by joule heat, which may influence the clinical outcomes. To establish the role of tissue heating in kHZ-SCS, a decisive first step is to characterize the range of temperature changes expected during conventional and kHZ-SCS protocols. We modeled a high-resolution CAD derived SCS model with detailed anatomy to predict the temperature increases by a SCS lead using a bioheat transfer multi-physics based on joule heat. Temperature increases were predicted in a closed tissue compartments (soft tissue, vertebral bone, epidural space (predominantly fat with minimal perfusion), intervertebral disc, meninges, spinal cord with nerve roots) of geometric human spinal cord model under varied parameterization. The experimentally constrained bio-heat model shows SCS waveform power (waveform RMS) determines tissue heating at the spinal cord and surrounding tissues. For example, we predict temperature increased at dorsal spinal cord of 0.56-1.04 °C during 3.5 mA peak 10 KHz stimulation with a 40-10-40 μs biphasic pulse pattern, 0.09-0.20 °C during 3.5 mA 1 KHz 100-100-100 μs stimulation, and 0.10 °C during 3.5 mA 50 Hz 200-100-200 μs stimulation. Notably, peak heating of the spinal cord and other tissues increases superlinearly with stimulation power and so are especially sensitive to incremental changes in SCS pulse amplitude or frequency (with associated pulse compression). Further supporting distinct SCS intervention strategies based on heating; the spatial profile of temperature changes is more uniform compared to electric fields, which suggests less sensitivity to lead position. Tissue heating may impact short and long-term outcomes of kHZ-SCS, and even as an adjunct mechanism, suggests distinct strategies for lead position and programming optimization.

Expression of the axon-guidance protein receptor Neuropilin 1 is increased in the spinal cord and decreased in muscle of a mouse model of amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a degenerative motor neuron disorder. It is supposed that ALS is at least in part an axonopathy. Neuropilin 1 is an important receptor of the axon repellent Semaphorin 3A and a co-receptor of vascular endothelial growth factor. It is probably involved in neuronal and axonal de-/regeneration and might be of high relevance for ALS pathogenesis and/or disease progression. To elucidate whether the expression of either Neuropilin1 or Semaphorin3A is altered in ALS we investigated these proteins in human brain, spinal cord and muscle tissue of ALS-patients and controls as well as transgenic SOD1G93A and control mice. Neuropilin1 and Semaphorin3A gene and protein expression were assessed by quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry. Groups were compared using either Student t-test or Mann-Whitney U test. We observed a consistent increase of Neuropilin1 expression in the spinal cord and decrease of Neuropilin1 and Semaphorin3A in muscle tissue of transgenic SOD1G93A mice at the mRNA and protein level. Previous studies have shown that damage of neurons physiologically causes Neuropilin1 and Semaphorin3A increase in the central nervous system and decrease in the peripheral nervous system. Our results indicate that this also occurs in ALS. Pharmacological modulation of expression and function of axon repellents could be a promising future therapeutic option in ALS.

Biomimetic 3D-printed scaffolds for spinal cord injury repair

Current methods for bioprinting functional tissue lack appropriate biofabrication techniques to build complex 3D microarchitectures essential for guiding cell growth and promoting tissue maturation1. 3D printing of central nervous system (CNS) structures has not been accomplished, possibly owing to the complexity of CNS architecture. Here, we report the use of a microscale continuous projection printing method (μCPP) to create a complex CNS structure for regenerative medicine applications in the spinal cord. μCPP can print 3D biomimetic hydrogel scaffolds tailored to the dimensions of the rodent spinal cord in 1.6 s and is scalable to human spinal cord sizes and lesion geometries. We tested the ability of µCPP 3D-printed scaffolds loaded with neural progenitor cells (NPCs) to support axon regeneration and form new 'neural relays' across sites of complete spinal cord injury in vivo in rodents1,2. We find that injured host axons regenerate into 3D biomimetic scaffolds and synapse onto NPCs implanted into the device and that implanted NPCs in turn extend axons out of the scaffold and into the host spinal cord below the injury to restore synaptic transmission and significantly improve functional outcomes. Thus, 3D biomimetic scaffolds offer a means of enhancing CNS regeneration through precision medicine.