RESUMEN
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 1â d, 7â d, and 8â 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 (8â weeks) after injury. TLR4 null mice also showed reduction of scar-related ECM molecules at 8â 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.
Asunto(s)
Citocinas , Traumatismos de la Médula Espinal , Ratones , Femenino , Animales , Citocinas/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Neuronas/metabolismo , Inflamación/metabolismo , Ratones Noqueados , Médula Espinal/metabolismo , Recuperación de la Función/fisiologíaRESUMEN
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.
Asunto(s)
Condroitina ABC Liasa , Traumatismos de la Médula Espinal , Animales , Condroitina ABC Liasa/farmacología , Femenino , Miembro Anterior/inervación , Miembro Anterior/fisiología , Músculo Esquelético , Ratas , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/terapia , Extremidad SuperiorRESUMEN
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.
Asunto(s)
Condroitina ABC Liasa/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Regeneración Nerviosa/genética , Raíces Nerviosas Espinales/fisiología , Animales , Axones/fisiología , Línea Celular , Femenino , Terapia Genética/métodos , Células HEK293 , Humanos , Neuronas Motoras/fisiología , Regeneración Nerviosa/fisiología , Ratas , Ratas Wistar , Recuperación de la Función/genética , Células de Schwann/fisiologíaRESUMEN
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.
Asunto(s)
Receptores ErbB/deficiencia , Células Precursoras de Oligodendrocitos/metabolismo , Recuperación de la Función/fisiología , Remielinización/fisiología , Transducción de Señal/fisiología , Traumatismos de la Médula Espinal/metabolismo , Animales , Receptores ErbB/genética , Ratones , Ratones Transgénicos , Células de Schwann/metabolismo , Traumatismos de la Médula Espinal/patologíaRESUMEN
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.
Asunto(s)
Condroitina ABC Liasa/administración & dosificación , Terapia Genética/métodos , Traumatismos de la Médula Espinal/tratamiento farmacológico , Animales , Condroitina ABC Liasa/farmacología , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Técnicas de Transferencia de Gen , Regeneración Nerviosa/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Ratas , Ratas Mutantes , Recuperación de la Función/fisiología , Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Transgenes/genéticaRESUMEN
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.
Asunto(s)
Biología Computacional , Perfilación de la Expresión Génica , Proteoma , Proteómica , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/metabolismo , Transcriptoma , Animales , Biología Computacional/métodos , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Proteómica/métodos , Ratas , Traumatismos de la Médula Espinal/tratamiento farmacológico , Factores de TiempoRESUMEN
Following traumatic spinal cord injury, acute demyelination of spinal axons is followed by a period of spontaneous remyelination. However, this endogenous repair response is suboptimal and may account for the persistently compromised function of surviving axons. Spontaneous remyelination is largely mediated by Schwann cells, where demyelinated central axons, particularly in the dorsal columns, become associated with peripheral myelin. The molecular control, functional role and origin of these central remyelinating Schwann cells is currently unknown. The growth factor neuregulin-1 (Nrg1, encoded by NRG1) is a key signalling factor controlling myelination in the peripheral nervous system, via signalling through ErbB tyrosine kinase receptors. Here we examined whether Nrg1 is required for Schwann cell-mediated remyelination of central dorsal column axons and whether Nrg1 ablation influences the degree of spontaneous remyelination and functional recovery following spinal cord injury. In contused adult mice with conditional ablation of Nrg1, we found an absence of Schwann cells within the spinal cord and profound demyelination of dorsal column axons. There was no compensatory increase in oligodendrocyte remyelination. Removal of peripheral input to the spinal cord and proliferation studies demonstrated that the majority of remyelinating Schwann cells originated within the injured spinal cord. We also examined the role of specific Nrg1 isoforms, using mutant mice in which only the immunoglobulin-containing isoforms of Nrg1 (types I and II) were conditionally ablated, leaving the type III Nrg1 intact. We found that the immunoglobulin Nrg1 isoforms were dispensable for Schwann cell-mediated remyelination of central axons after spinal cord injury. When functional effects were examined, both global Nrg1 and immunoglobulin-specific Nrg1 mutants demonstrated reduced spontaneous locomotor recovery compared to injured controls, although global Nrg1 mutants were more impaired in tests requiring co-ordination, balance and proprioception. Furthermore, electrophysiological assessments revealed severely impaired axonal conduction in the dorsal columns of global Nrg1 mutants (where Schwann cell-mediated remyelination is prevented), but not immunoglobulin-specific mutants (where Schwann cell-mediated remyelination remains intact), providing robust evidence that the profound demyelinating phenotype observed in the dorsal columns of Nrg1 mutant mice is related to conduction failure. Our data provide novel mechanistic insight into endogenous regenerative processes after spinal cord injury, demonstrating that Nrg1 signalling regulates central axon remyelination and functional repair and drives the trans-differentiation of central precursor cells into peripheral nervous system-like Schwann cells that remyelinate spinal axons after injury. Manipulation of the Nrg1 system could therefore be exploited to enhance spontaneous repair after spinal cord injury and other central nervous system disorders with a demyelinating pathology.media-1vid110.1093/brain/aww039_video_abstractaww039_video_abstract.
Asunto(s)
Vaina de Mielina/fisiología , Neurregulina-1/fisiología , Recuperación de la Función/fisiología , Células de Schwann/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Regeneración de la Medula Espinal/fisiología , Animales , Axones/fisiología , Axones/ultraestructura , Proliferación Celular , Enfermedades Desmielinizantes/fisiopatología , Femenino , Ratones , Ratones Mutantes , Destreza Motora/fisiología , Vaina de Mielina/ultraestructura , Conducción Nerviosa/fisiología , Neurregulina-1/biosíntesis , Neurregulina-1/genética , Isoformas de Proteínas/fisiología , Ratas , Recuperación de la Función/genética , Médula Espinal/metabolismo , Médula Espinal/fisiopatología , Médula Espinal/ultraestructura , Traumatismos de la Médula Espinal/genéticaRESUMEN
Chondroitinase ABC (ChABC) has striking effects on promoting neuronal plasticity after spinal cord injury (SCI), but little is known about its involvement in other pathological mechanisms. Recent work showed that ChABC might also modulate the immune response by promoting M2 macrophage polarization. Here we investigate in detail the immunoregulatory effects of ChABC after SCI in rats. Initially, we examined the expression profile of 16 M1/M2 macrophage polarization markers at 3 h and 7 d postinjury. ChABC treatment had a clear effect on the immune signature after SCI. More specifically, ChABC increased the expression of the anti-inflammatory cytokine IL-10, accompanied by a reduction in the proinflammatory cytokine IL-12B in injured spinal tissue. These effects were associated with a distinct, IL-10-mediated anti-inflammatory response in ChABC-treated spinal cords. Mechanistically, we show that IL-10 expression is driven by tissue injury and macrophage infiltration, while the p38 MAPK is the central regulator of IL-10 expression in vivo. These findings provide novel insights into the effects of ChABC in the injured spinal cord and explain its immunoregulatory activity.
Asunto(s)
Condroitina ABC Liasa/fisiología , Regulación de la Expresión Génica , Inmunomodulación/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Interleucina-10/biosíntesis , Traumatismos de la Médula Espinal/inmunología , Animales , Condroitina ABC Liasa/administración & dosificación , Condroitina ABC Liasa/farmacología , Imidazoles/farmacología , Inmunomodulación/fisiología , Inyecciones Espinales , Interleucina-12/biosíntesis , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/fisiología , Masculino , Proteoglicanos/metabolismo , Piridinas/farmacología , Ratas , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
Chondroitin sulfate proteoglycans (CSPGs) inhibit repair following spinal cord injury. Here we use mammalian-compatible engineered chondroitinase ABC (ChABC) delivered via lentiviral vector (LV-ChABC) to explore the consequences of large-scale CSPG digestion for spinal cord repair. We demonstrate significantly reduced secondary injury pathology in adult rats following spinal contusion injury and LV-ChABC treatment, with reduced cavitation and enhanced preservation of spinal neurons and axons at 12 weeks postinjury, compared with control (LV-GFP)-treated animals. To understand these neuroprotective effects, we investigated early inflammatory changes following LV-ChABC treatment. Increased expression of the phagocytic macrophage marker CD68 at 3 d postinjury was followed by increased CD206 expression at 2 weeks, indicating that large-scale CSPG digestion can alter macrophage phenotype to favor alternatively activated M2 macrophages. Accordingly, ChABC treatment in vitro induced a significant increase in CD206 expression in unpolarized monocytes stimulated with conditioned medium from spinal-injured tissue explants. LV-ChABC also promoted the remodelling of specific CSPGs as well as enhanced vascularity, which was closely associated with CD206-positive macrophages. Neuroprotective effects of LV-ChABC corresponded with improved sensorimotor function, evident as early as 1 week postinjury, a time point when increased neuronal survival correlated with reduced apoptosis. Improved function was maintained into chronic injury stages, where improved axonal conduction and increased serotonergic innervation were also observed. Thus, we demonstrate that ChABC gene therapy can modulate secondary injury processes, with neuroprotective effects that lead to long-term improved functional outcome and reveal novel mechanistic evidence that modulation of macrophage phenotype may underlie these effects.
Asunto(s)
Condroitina ABC Liasa/genética , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Terapia Genética/métodos , Macrófagos/fisiología , Traumatismos de la Médula Espinal/terapia , Animales , Células Cultivadas , Proteoglicanos Tipo Condroitín Sulfato/administración & dosificación , Modelos Animales de Enfermedad , Estimulación Eléctrica , Femenino , Regulación de la Expresión Génica/fisiología , Inyecciones Espinales , Proteínas del Tejido Nervioso/metabolismo , Conducción Nerviosa/efectos de los fármacos , Conducción Nerviosa/fisiología , Desempeño Psicomotor/fisiología , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Factores de TiempoRESUMEN
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.
Asunto(s)
Células Madre Embrionarias Humanas , Humanos , Diferenciación Celular , Proteínas Hedgehog/metabolismo , Interneuronas/metabolismo , Neuronas Motoras/metabolismo , Médula Espinal/metabolismo , Proteína Homeobox Nkx-2.2/genéticaRESUMEN
In the majority of spinal cord injuries (SCIs), some axonal projections remain intact. We examined the functional status of these surviving axons since they represent a prime therapeutic target. Using a novel electrophysiological preparation, adapted from techniques used to study primary demyelination, we quantified conduction failure across a SCI and studied conduction changes over time in adult rats with a moderate severity spinal contusion (150 kdyn; Infinite Horizon impactor). By recording antidromically activated single units from teased dorsal root filaments, we demonstrate complete conduction block in ascending dorsal column axons acutely (1-7 d) after injury, followed by a period of restored conduction over the subacute phase (2-4 weeks), with no further improvements in conduction at chronic stages (3-6 months). By cooling the lesion site, additional conducting fibers could be recruited, thus revealing a population of axons that are viable but unable to conduct under normal physiological conditions. Importantly, this phenomenon is still apparent at the most chronic (6 month) time point. The time course of conduction changes corresponded with changes in behavioral function, and ultrastructural analysis of dorsal column axons revealed extensive demyelination during the period of conduction block, followed by progressive remyelination. A proportion of dorsal column axons remained chronically demyelinated, suggesting that these are the axons recruited with the cooling paradigm. Thus, using a clinically relevant SCI model, we have identified a population of axons present at chronic injury stages that are intact but fail to conduct and are therefore a prime target for therapeutic strategies to restore function.
Asunto(s)
Axones/fisiología , Conducción Nerviosa/fisiología , Neuronas/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Médula Espinal/fisiopatología , Potenciales de Acción/fisiología , Animales , Femenino , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Chondroitin sulphate proteoglycans (CSPGs) are extracellular matrix molecules whose inhibitory activity is attenuated by the enzyme chondroitinase ABC (ChABC). Here we assess whether CSPG degradation can promote compensatory sprouting of the intact corticospinal tract (CST) following unilateral injury and restore function to the denervated forelimb. Adult C57BL/6 mice underwent unilateral pyramidotomy and treatment with either ChABC or a vehicle control. Significant impairments in forepaw symmetry were observed following pyramidotomy, with injured mice preferentially using their intact paw during spontaneous vertical exploration of a cylinder. No recovery on this task was observed in vehicle-treated mice. However, ChABC-treated mice showed a marked recovery of function, with forelimb symmetry fully restored by 5 weeks post-injury. Functional recovery was associated with robust sprouting of the uninjured CST, with numerous axons observed crossing the midline in the brainstem and spinal cord and terminating in denervated grey matter. CST fibres in the denervated side of the spinal cord following ChABC treatment were closely associated with the synaptic marker vGlut1. Immunohistochemical assessment of chondroitin-4-sulphate revealed that CSPGs were heavily digested around lamina X, alongside midline crossing axons and in grey matter regions where sprouting axons and reduced peri-neuronal net staining was observed. Thus, we demonstrate that CSPG degradation promotes midline crossing and reinnervation of denervated target regions by intact CST axons and leads to restored function in the denervated forepaw. Enhancing compensatory sprouting using ChABC provides a route to restore function that could be applied to disorders such as spinal cord injury and stroke.
Asunto(s)
Condroitina ABC Liasa/farmacología , Miembro Anterior/inervación , Tractos Piramidales/fisiopatología , Animales , Axones/efectos de los fármacos , Axones/patología , Condroitina ABC Liasa/metabolismo , Condroitina ABC Liasa/uso terapéutico , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Sulfatos de Condroitina/metabolismo , Desnervación , Masculino , Ratones , Ratones Endogámicos C57BL , Tractos Piramidales/patología , Tractos Piramidales/cirugía , Traumatismos de la Médula Espinal/tratamiento farmacológico , Regeneración de la Medula Espinal/efectos de los fármacosRESUMEN
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.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato , Traumatismos de la Médula Espinal , Animales , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Inflamación , Roedores , Traumatismos de la Médula Espinal/patología , Receptor Toll-Like 4/genéticaRESUMEN
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.
Asunto(s)
Contusiones/diagnóstico por imagen , Traumatismos de la Médula Espinal/diagnóstico por imagen , Sincrotrones , Microtomografía por Rayos X/métodos , Animales , Edema/etiología , Edema/patología , Sustancia Gris/diagnóstico por imagen , Fuerza de la Mano , Imagenología Tridimensional , Masculino , Adhesión en Parafina , Ratas , Médula Espinal/irrigación sanguínea , Médula Espinal/diagnóstico por imagen , Fijación del Tejido , Sustancia Blanca/diagnóstico por imagenRESUMEN
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.
Asunto(s)
Células-Madre Neurales , Traumatismos de la Médula Espinal , Animales , Axones , Condroitina ABC Liasa/farmacología , Proteoglicanos Tipo Condroitín Sulfato , Regeneración Nerviosa , Células-Madre Neurales/trasplante , Ratas , Médula Espinal , Traumatismos de la Médula Espinal/terapiaRESUMEN
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.
Asunto(s)
Sistema Nervioso Central/metabolismo , Condroitina ABC Liasa/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Sistema Nervioso Periférico/metabolismo , Animales , Astrocitos/metabolismo , Axones/metabolismo , Adhesión Celular , Movimiento Celular , Células Cultivadas , Femenino , Terapia Genética , Integrinas/metabolismo , Lentivirus/enzimología , Regeneración Nerviosa/efectos de los fármacos , Neuritas/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Células de Schwann/metabolismo , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
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.
Asunto(s)
Bienestar del Animal/normas , Modelos Animales de Enfermedad , Traumatismos de la Médula Espinal , Animales , RoedoresRESUMEN
Chondroitinase ABC (ChABC) represents a promising therapeutic strategy for the treatment of spinal cord injury due to its potent effects on restoring function to spinal-injured adult mammals. However, there is limited mechanistic insight as to the underlying effects of ChABC treatment, where the effects are mediated, and which signaling pathways are involved in ChABC-mediated repair. Here we use a transgenic (YFP-H) mouse to demonstrate that cortical layer V projection neurons undergo severe atrophy 4 weeks after thoracic dorsal column injury and that ChABC is neuroprotective for these neurons after ICV infusion. ChABC also prevented cell atrophy after localized delivery to the spinal cord, suggesting a possible retrograde neuroprotective effect mediated at the injury site. Furthermore, neuroprotection of corticospinal cell somata coincided with increased axonal sprouting in the spinal cord. In addition, Western blot analysis of a number of kinases important in survival and growth signaling revealed a significant increase in phosphorylated ERK1 at the spinal injury site after in vivo ChABC treatment, indicating that activated ERK may play a role in downstream repair processes after ChABC treatment. Total forms of PKC and AKT were also elevated, indicating that modification of the glial scar by ChABC promotes long-lasting signaling changes at the lesion site. Thus, using the YFP-H mouse as a novel tool to study degenerative changes and repair after spinal cord injury we demonstrate, for the first time, that ChABC treatment regulates multiple signaling cascades at the injury site and exerts protective effects on axotomized corticospinal projection neurons.