Tlr4 Deletion Modulates Cytokine and Extracellular Matrix Expression in Chronic Spinal Cord Injury, Leading to Improved Secondary Damage and Functional Recovery.

Toll-like receptors (TLRs) play an important role in the innate immune response after CNS injury. Although TLR4 is one of the best characterized, its role in chronic stages after spinal cord injury (SCI) is not well understood. We examined the role of TLR4 signaling in injury-induced responses at 1 d, 7 d, and 8 weeks after spinal cord contusion injury in adult female TLR4 null and wild-type mice. Analyses include secondary damage, a range of transcriptome and protein analyses of inflammatory, cell death, and extracellular matrix (ECM) molecules, as well as immune cell infiltration and changes in axonal sprouting and locomotor recovery. Lack of TLR4 signaling results in reduced neuronal and myelin loss, reduced activation of NFκB, and decreased expression of inflammatory cytokines and necroptotic cell death pathway at a late time point (8 weeks) after injury. TLR4 null mice also showed reduction of scar-related ECM molecules at 8 weeks after SCI, accompanied by increase in ECM molecules associated with perineuronal nets, increased sprouting of serotonergic fibers, and improved locomotor recovery. These findings reveal novel effects of TLR4 signaling in chronic SCI. We show that TLR4 influences inflammation, cell death, and ECM deposition at late-stage post-injury when secondary injury processes are normally considered to be over. This highlights the potential for late-stage targeting of TLR4 as a potential therapy for chronic SCI.

Enrichment of human embryonic stem cell-derived V3 interneurons using an Nkx2-2 gene-specific reporter.

V3 spinal interneurons are a key element of the spinal circuits, which control motor function. However, to date, there are no effective ways of deriving a pure V3 population from human pluripotent stem cells. Here, we report a method for differentiation and isolation of spinal V3 interneurons, combining extrinsic factor-mediated differentiation and magnetic activated cell sorting. We found that differentiation of V3 progenitors can be enhanced with a higher concentration of Sonic Hedgehog agonist, as well as culturing cells in 3D format. To enable V3 progenitor purification from mixed differentiation cultures, we developed a transgene reporter, with a part of the regulatory region of V3-specific gene Nkx2-2 driving the expression of a membrane marker CD14. We found that in human cells, NKX2-2 initially exhibited co-labelling with motor neuron progenitor marker, but V3 specificity emerged as the differentiation culture progressed. At these later differentiation timepoints, we were able to enrich V3 progenitors labelled with CD14 to ~ 95% purity, and mature them to postmitotic V3 interneurons. This purification tool for V3 interneurons will be useful for in vitro disease modeling, studies of normal human neural development and potential cell therapies for disorders of the spinal cord.

Chronic muscle recordings reveal recovery of forelimb function in spinal injured female rats after cortical epidural stimulation combined with rehabilitation and chondroitinase ABC.

Cervical level spinal cord injury (SCI) can severely impact upper limb muscle function, which is typically assessed in the clinic using electromyography (EMG). Here, we established novel preclinical methodology for EMG assessments of muscle function after SCI in awake freely moving animals. Adult female rats were implanted with EMG recording electrodes in bicep muscles and received bilateral cervical (C7) contusion injuries. Forelimb muscle activity was assessed by recording maximum voluntary contractions during a grip strength task and cortical motor evoked potentials in the biceps. We demonstrate that longitudinal recordings of muscle activity in the same animal are feasible over a chronic post-injury time course and provide a sensitive method for revealing post-injury changes in muscle activity. This methodology was utilized to investigate recovery of muscle function after a novel combination therapy. Cervical contused animals received intraspinal injections of a neuroplasticity-promoting agent (lentiviral-chondroitinase ABC) plus 11 weeks of cortical epidural electrical stimulation (3 h daily, 5 days/week) and behavioral rehabilitation (15 min daily, 5 days/week). Longitudinal monitoring of voluntary and evoked muscle activity revealed significantly increased muscle activity and upper limb dexterity with the combination treatment, compared to a single treatment or no treatment. Retrograde mapping of motor neurons innervating the biceps showed a predominant distribution across spinal segments C5-C8, indicating that treatment effects were likely due to neuroplastic changes in a mixture of intact and injured motor neurons. Thus, longitudinal assessments of muscle function after SCI correlate with skilled reach and grasp performance and reveal functional benefits of a novel combination therapy.

Chondroitin sulfate proteoglycans prevent immune cell phenotypic conversion and inflammation resolution via TLR4 in rodent models of spinal cord injury.

Chondroitin sulfate proteoglycans (CSPGs) act as potent inhibitors of axonal growth and neuroplasticity after spinal cord injury (SCI). Here we reveal that CSPGs also play a critical role in preventing inflammation resolution by blocking the conversion of pro-inflammatory immune cells to a pro-repair phenotype in rodent models of SCI. We demonstrate that enzymatic digestion of CSPG glycosaminoglycans enhances immune cell clearance and reduces pro-inflammatory protein and gene expression profiles at key resolution time points. Analysis of phenotypically distinct immune cell clusters revealed CSPG-mediated modulation of macrophage and microglial subtypes which, together with T lymphocyte infiltration and composition changes, suggests a role for CSPGs in modulating both innate and adaptive immune responses after SCI. Mechanistically, CSPG activation of a pro-inflammatory phenotype in pro-repair immune cells was found to be TLR4-dependent, identifying TLR4 signalling as a key driver of CSPG-mediated immune modulation. These findings establish CSPGs as critical mediators of inflammation resolution failure after SCI in rodents, which leads to prolonged inflammatory pathology and irreversible tissue destruction.

Combined treatment with enteric neural stem cells and chondroitinase ABC reduces spinal cord lesion pathology.

BACKGROUND: Spinal cord injury (SCI) presents a significant challenge for the field of neurotherapeutics. Stem cells have shown promise in replenishing the cells lost to the injury process, but the release of axon growth-inhibitory molecules such as chondroitin sulfate proteoglycans (CSPGs) by activated cells within the injury site hinders the integration of transplanted cells. We hypothesised that simultaneous application of enteric neural stem cells (ENSCs) isolated from the gastrointestinal tract, with a lentivirus (LV) containing the enzyme chondroitinase ABC (ChABC), would enhance the regenerative potential of ENSCs after transplantation into the injured spinal cord. METHODS: ENSCs were harvested from the GI tract of p7 rats, expanded in vitro and characterised. Adult rats bearing a contusion injury were randomly assigned to one of four groups: no treatment, LV-ChABC injection only, ENSC transplantation only or ENSC transplantation+LV-ChABC injection. After 16 weeks, rats were sacrificed and the harvested spinal cords examined for evidence of repair. RESULTS: ENSC cultures contained a variety of neuronal subtypes suitable for replenishing cells lost through SCI. Following injury, transplanted ENSC-derived cells survived and ChABC successfully degraded CSPGs. We observed significant reductions in the injured tissue and cavity area, with the greatest improvements seen in the combined treatment group. ENSC-derived cells extended projections across the injury site into both the rostral and caudal host spinal cord, and ENSC transplantation significantly increased the number of cells extending axons across the injury site. Furthermore, the combined treatment resulted in a modest, but significant functional improvement by week 16, and we found no evidence of the spread of transplanted cells to ectopic locations or formation of tumours. CONCLUSIONS: Regenerative effects of a combined treatment with ENSCs and ChABC surpassed either treatment alone, highlighting the importance of further research into combinatorial therapies for SCI. Our work provides evidence that stem cells taken from the adult gastrointestinal tract, an easily accessible source for autologous transplantation, could be strongly considered for the repair of central nervous system disorders.

The spatiotemporal spread of cervical spinal cord contusion injury pathology revealed by 3D in-line phase contrast synchrotron X-ray microtomography.

Extensive structural changes occur within the spinal cord following traumatic injury. Acute tissue debris and necrotic tissue are broken down, proliferating local glia and infiltrating leukocytes remodel tissue biochemical and biophysical properties, and a chronic cavity surrounded by a scar forms at the injury epicentre. Serial-section 2D histology has traditionally assessed these features in experimental models of spinal cord injury (SCI) to measure the extent of tissue pathology and evaluate efficacy of novel therapies. However, this 2D snapshot approach overlooks slice intervening features, with accurate representation of tissue compromised by mechanical processing artefacts. 3D imaging avoids these caveats and allows full exploration of the injured tissue volume to characterise whole tissue pathology. Amongst 3D imaging modalities, Synchrotron Radiation X-ray microtomography (SRµCT) is advantageous for its speed, ability to cover large tissue volumes at high resolution, and need for minimal sample processing. Here we demonstrate how extended lengths of formalin-fixed, paraffin-embedded (FFPE) rat spinal cord can be completely imaged by SRµCT with micron resolution. Label-free contrast derived from X-ray phase interactions with low-density soft tissues, reveals spinal cord white matter, gray matter, tissue damage and vasculature, with tissue still viable for targeted 2D-histology after 3D imaging. We used SRµCT to quantify tissue pathology after a midline, cervical level (C6), 225 kDyne contusion injury over acute-to-chronic (24 h to 5 weeks) post injury time points. Quantification revealed acute tissue swelling prior to chronic atrophy across the whole imaged region (spanning 2 spinal segments above and below injury), along with rostro-caudal asymmetries in white and gray matter volume loss. 3D volumes revealed satellite damage in tissue far removed from the epicentre, and extensive rostro-caudal spread of damage through the base of the dorsal columns at 24 h post injury. This damage overlapped regions of vasogenic oedema, confirmed with subsequent histology. Tissue damage at later time points in border regions was most prominent in the dorsal columns, where it overlapped sites of damaged venous vasculature. Elaborating rostro-caudal and spatiotemporal asymmetries in reduced traumatic injury models centred on these regions may inform future treatments that seek to limit the spread of tissue pathology to these 'at-risk' regions.

Secretion of a mammalian chondroitinase ABC aids glial integration at PNS/CNS boundaries.

Schwann cell grafts support axonal growth following spinal cord injury, but a boundary forms between the implanted cells and host astrocytes. Axons are reluctant to exit the graft tissue in large part due to the surrounding inhibitory environment containing chondroitin sulphate proteoglycans (CSPGs). We use a lentiviral chondroitinase ABC, capable of being secreted from mammalian cells (mChABC), to examine the repercussions of CSPG digestion upon Schwann cell behaviour in vitro. We show that mChABC transduced Schwann cells robustly secrete substantial quantities of the enzyme causing large-scale CSPG digestion, facilitating the migration and adhesion of Schwann cells on inhibitory aggrecan and astrocytic substrates. Importantly, we show that secretion of the engineered enzyme can aid the intermingling of cells at the Schwann cell-astrocyte boundary, enabling growth of neurites over the putative graft/host interface. These data were echoed in vivo. This study demonstrates the profound effect of the enzyme on cellular motility, growth and migration. This provides a cellular mechanism for mChABC induced functional and behavioural recovery shown in in vivo studies. Importantly, we provide in vitro evidence that mChABC gene therapy is equally or more effective at producing these effects as a one-time application of commercially available ChABC.

Combining timed GDNF and ChABC gene therapy to promote long-distance regeneration following ventral root avulsion and repair.

Ventral root avulsion leads to severe motoneuron degeneration and prolonged distal nerve denervation. After a critical period, a state of chronic denervation develops as repair Schwann cells lose their pro-regenerative properties and inhibitory factors such as CSPGs accumulate in the denervated nerve. In rats with ventral root avulsion injuries, we combined timed GDNF gene therapy delivered to the proximal nerve roots with the digestion of inhibitory CSPGs in the distal denervated nerve using sustained lentiviral-mediated chondroitinase ABC (ChABC) enzyme expression. Following reimplantation of lumbar ventral roots, timed GDNF-gene therapy enhanced motoneuron survival up to 45 weeks and improved axonal outgrowth, electrophysiological recovery, and muscle reinnervation. Despite a timed GDNF expression period, a subset of animals displayed axonal coils. Lentiviral delivery of ChABC enabled digestion of inhibitory CSPGs for up to 45 weeks in the chronically denervated nerve. ChABC gene therapy alone did not enhance motoneuron survival, but led to improved muscle reinnervation and modest electrophysiological recovery during later stages of the regeneration process. Combining GDNF treatment with digestion of inhibitory CSPGs did not have a significant synergistic effect. This study suggests a delicate balance exists between treatment duration and concentration in order to achieve therapeutic effects.

Refining rodent models of spinal cord injury.

This report was produced by an Expert Working Group (EWG) consisting of UK-based researchers, veterinarians and regulators of animal experiments with specialist knowledge of the use of animal models of spinal cord injury (SCI). It aims to facilitate the implementation of the Three Rs (Replacement, Reduction and Refinement), with an emphasis on refinement. Specific animal welfare issues were identified and discussed, and practical measures proposed, with the aim of reducing animal use and suffering, reducing experimental variability, and increasing translatability within this critically important research field.

Moving beyond the glial scar for spinal cord repair.

Traumatic spinal cord injury results in severe and irreversible loss of function. The injury triggers a complex cascade of inflammatory and pathological processes, culminating in formation of a scar. While traditionally referred to as a glial scar, the spinal injury scar in fact comprises multiple cellular and extracellular components. This multidimensional nature should be considered when aiming to understand the role of scarring in limiting tissue repair and recovery. In this Review we discuss recent advances in understanding the composition and phenotypic characteristics of the spinal injury scar, the oversimplification of defining the scar in binary terms as good or bad, and the development of therapeutic approaches to target scar components to enable improved functional outcome after spinal cord injury.

ErbB receptor signaling directly controls oligodendrocyte progenitor cell transformation and spontaneous remyelination after spinal cord injury.

We recently discovered a novel role for neuregulin-1 (Nrg1) signaling in mediating spontaneous regenerative processes and functional repair after spinal cord injury (SCI). We revealed that Nrg1 is the molecular signal responsible for spontaneous functional remyelination of dorsal column axons by peripheral nervous system (PNS)-like Schwann cells after SCI. Here, we investigate whether Nrg1/ErbB signaling controls the unusual transformation of centrally derived progenitor cells into these functional myelinating Schwann cells after SCI using a fate-mapping/lineage tracing approach. Specific ablation of Nrg1-ErbB receptors in central platelet-derived growth factor receptor alpha (PDGFRα)-derived lineage cells (using PDGFRαCreERT2/Tomato-red reporter mice crossed with ErbB3fl/fl/ErbB4fl/fl mice) led to a dramatic reduction in P0-positive remyelination in the dorsal columns following spinal contusion injury. Central myelination, assessed by Olig2 and proteolipid protein expression, was unchanged. Loss of ErbB signaling in PDGFRα lineage cells also significantly impacted the degree of spontaneous locomotor recovery after SCI, particularly in tests dependent on proprioception. These data have important implications, namely (a) cells from the PDGFRα-expressing progenitor lineage (which are presumably oligodendrocyte progenitor cells, OPCs) can differentiate into remyelinating PNS-like Schwann cells after traumatic SCI, (b) this process is controlled by ErbB tyrosine kinase signaling, and (c) this endogenous repair mechanism has significant consequences for functional recovery after SCI. Thus, ErbB tyrosine kinase receptor signaling directly controls the transformation of OPCs from the PDGFRα-expressing lineage into PNS-like functional remyelinating Schwann cells after SCI.

Neuromodulation in the restoration of function after spinal cord injury.

Neuromodulation, the use of electrical interfaces to alter neuronal activity, has been successful as a treatment approach in several neurological disorders, including deep brain stimulation for Parkinson's disease and epidural spinal stimulation for chronic pain. Neuromodulation can also be beneficial for spinal cord injury, from assisting basic functions such as respiratory pacing and bladder control, through to restoring volitional movements and skilled hand function. Approaches range from electrical stimulation of peripheral muscles, either directly or via brain-controlled bypass devices, to stimulation of the spinal cord and brain. Limitations to widespread clinical application include durability of neuromodulation devices, affordability and accessibility of some approaches, and poor understanding of the underlying mechanisms. Efforts to overcome these challenges through advances in technology, together with pragmatic knowledge gained from clinical trials and basic research, could lead to personalised neuromodulatory interventions to meet the specific needs of individuals with spinal cord injury.

Optimising complementary soft tissue synchrotron X-ray microtomography for reversibly-stained central nervous system samples.

Synchrotron radiation microtomography (SRµCT) is a nominally non-destructive 3D imaging technique which can visualise the internal structures of whole soft tissues. As a multi-stage technique, the cumulative benefits of optimising sample preparation, scanning parameters and signal processing can improve SRµCT imaging efficiency, image quality, accuracy and ultimately, data utility. By evaluating different sample preparations (embedding media, tissue stains), imaging (projection number, propagation distance) and reconstruction (artefact correction, phase retrieval) parameters, a novel methodology (combining reversible iodine stain, wax embedding and inline phase contrast) was optimised for fast (~12 minutes), high-resolution (3.2-4.8 µm diameter capillaries resolved) imaging of the full diameter of a 3.5 mm length of rat spinal cord. White-grey matter macro-features and micro-features such as motoneurons and capillary-level vasculature could then be completely segmented from the imaged volume for analysis through the shallow machine learning SuRVoS Workbench. Imaged spinal cord tissue was preserved for subsequent histology, establishing a complementary SRµCT methodology that can be applied to study spinal cord pathologies or other nervous system tissues such as ganglia, nerves and brain. Further, our 'single-scan iterative downsampling' approach and side-by-side comparisons of mounting options, sample stains and phase contrast parameters should inform efficient, effective future soft tissue SRµCT experiment design.

Immune-evasive gene switch enables regulated delivery of chondroitinase after spinal cord injury.

Chondroitinase ABC is a promising preclinical therapy that promotes functional neuroplasticity after CNS injury by degrading extracellular matrix inhibitors. Efficient delivery of chondroitinase ABC to the injured mammalian spinal cord can be achieved by viral vector transgene delivery. This approach dramatically modulates injury pathology and restores sensorimotor functions. However, clinical development of this therapy is limited by a lack of ability to exert control over chondroitinase gene expression. Prior experimental gene regulation platforms are likely to be incompatible with the non-resolving adaptive immune response known to occur following spinal cord injury. Therefore, here we apply a novel immune-evasive dual vector system, in which the chondroitinase gene is under a doxycycline inducible regulatory switch, utilizing a chimeric transactivator designed to evade T cell recognition. Using this novel vector system, we demonstrate tight temporal control of chondroitinase ABC gene expression, effectively removing treatment upon removal of doxycycline. This enables a comparison of short and long-term gene therapy paradigms in the treatment of clinically-relevant cervical level contusion injuries in adult rats. We reveal that transient treatment (2.5 weeks) is sufficient to promote improvement in sensory axon conduction and ladder walking performance. However, in tasks requiring skilled reaching and grasping, only long term treatment (8 weeks) leads to significantly improved function, with rats able to accurately grasp and retrieve sugar pellets. The late emergence of skilled hand function indicates enhanced neuroplasticity and connectivity and correlates with increased density of vGlut1+ innervation in spinal cord grey matter, particularly in lamina III-IV above and below the injury. Thus, our novel gene therapy system provides an experimental tool to study temporal effects of extracellular matrix digestion as well as an encouraging step towards generating a safer chondroitinase gene therapy strategy, longer term administration of which increases neuroplasticity and recovery of descending motor control. This preclinical study could have a significant impact for tetraplegic individuals, for whom recovery of hand function is an important determinant of independence, and supports the ongoing development of chondroitinase gene therapy towards clinical application for the treatment of spinal cord injury.

Combined Transcriptomics, Proteomics and Bioinformatics Identify Drug Targets in Spinal Cord Injury.

Spinal cord injury (SCI) causes irreversible tissue damage and severe loss of neurological function. Currently, there are no approved treatments and very few therapeutic targets are under investigation. Here, we combined 4 high-throughput transcriptomics and proteomics datasets, 7 days and 8 weeks following clinically-relevant rat SCI to identify proteins with persistent differential expression post-injury. Out of thousands of differentially regulated entities our combined analysis identified 40 significantly upregulated versus 48 significantly downregulated molecules, which were persistently altered at the mRNA and protein level, 7 days and 8 weeks post-SCI. Bioinformatics analysis was then utilized to identify currently available drugs with activity against the filtered molecules and to isolate proteins with known or unknown function in SCI. Our findings revealed multiple overlooked therapeutic candidates with important bioactivity and established druggability but with unknown expression and function in SCI including the upregulated purine nucleoside phosphorylase (PNP), cathepsins A, H, Z (CTSA, CTSH, CTSZ) and proteasome protease PSMB10, as well as the downregulated ATP citrate lyase (ACLY), malic enzyme (ME1) and sodium-potassium ATPase (ATP1A3), amongst others. This work reveals previously unappreciated therapeutic candidates for SCI and available drugs, thus providing a valuable resource for further studies and potential repurposing of existing therapeutics for SCI.

Structural and Functional Substitution of Deleted Primary Sensory Neurons by New Growth from Intrinsic Spinal Cord Nerve Cells: An Alternative Concept in Reconstruction of Spinal Cord Circuits.

In a recent clinical report, return of the tendon stretch reflex was demonstrated after spinal cord surgery in a case of total traumatic brachial plexus avulsion injury. Peripheral nerve grafts had been implanted into the spinal cord to reconnect to the peripheral nerves for motor and sensory function. The dorsal root ganglia (DRG) containing the primary sensory nerve cells had been surgically removed in order for secondary or spinal cord sensory neurons to extend into the periphery and replace the deleted DRG neurons. The present experimental study uses a rat injury model first to corroborate the clinical finding of a re-established spinal reflex arch, and second, to elucidate some of the potential mechanisms underlying these findings by means of morphological, immunohistochemical, and electrophysiological assessments. Our findings indicate that, after spinal cord surgery, the central nervous system sensory system could replace the traumatically detached original peripheral sensory connections through new neurite growth from dendrites.

The soft mechanical signature of glial scars in the central nervous system.

Injury to the central nervous system (CNS) alters the molecular and cellular composition of neural tissue and leads to glial scarring, which inhibits the regrowth of damaged axons. Mammalian glial scars supposedly form a chemical and mechanical barrier to neuronal regeneration. While tremendous effort has been devoted to identifying molecular characteristics of the scar, very little is known about its mechanical properties. Here we characterize spatiotemporal changes of the elastic stiffness of the injured rat neocortex and spinal cord at 1.5 and three weeks post-injury using atomic force microscopy. In contrast to scars in other mammalian tissues, CNS tissue significantly softens after injury. Expression levels of glial intermediate filaments (GFAP, vimentin) and extracellular matrix components (laminin, collagen IV) correlate with tissue softening. As tissue stiffness is a regulator of neuronal growth, our results may help to understand why mammalian neurons do not regenerate after injury.