Role of melatonin in the dynamics of acute spinal cord injury in rats

Melatonin is well-documented to have the ability of reducing nerve inflammation and scavenging free radicals. However, the therapeutic effect of melatonin on spinal cord injury has not been fully described. In this study, we assessed the effect of melatonin on T9 spinal cord injury established by Allen method in rats. Melatonin deficiency significantly delayed the recovery of sensory and motor functions in SCI rats. Treatment with melatonin significantly alleviated neuronal apoptosis and accelerated the recovery of spinal cord function. These results suggest that melatonin is effective to ameliorate spinal cord injury through inhibition of neuronal apoptosis and promotion of neuronal repair.     

Involvement of acidic fibroblast growth factor in spinal cord injury repair processes revealed by a proteomics approach

Acidic fibroblast growth factor (aFGF; also known as FGF-1) is a potent neurotrophic factor that affects neuronal survival in the injured spinal cord. However, the pathological changes that occur with spinal cord injury (SCI) and the attribution to aFGF of a neuroprotective effect during SCI are still elusive. In this study, we demonstrated that rat SCI, when treated with aFGF, showed significant functional recovery as indicated by the Basso, Beattie, and Bresnahan locomotor rating scale and the combined behavior score (p < 0.01-0.001). Furthermore proteomics and bioinformatics approaches were adapted to investigate changes in the global protein profile of the damaged spinal cord tissue when experimental rats were treated either with or without aFGF at 24 h after injury. We found that 51 protein spots, resolvable by two-dimensional PAGE, had significant differential expression. Using hierarchical clustering analysis, these proteins were categorized into five major expression patterns. Noticeably proteins involved in the process of secondary injury, such as astrocyte activation (glial fibrillary acidic protein), inflammation (S100B), and scar formation (keratan sulfate proteoglycan lumican), which lead to the blocking of injured spinal cord regeneration, were down-regulated in the contusive spinal cord after treatment with aFGF. We propose that aFGF might initiate a series of biological processes to prevent or attenuate secondary injury and that this, in turn, leads to an improvement in functional recovery. Moreover the quantitative expression level of these proteins was verified by quantitative real time PCR. Furthermore we identified various potential neuroprotective protein factors that are induced by aFGF and may be involved in the spinal cord repair processes of SCI rats. Thus, our results could have a remarkable impact on clinical developments in the area of spinal cord injury therapy.     

Examination of the Combined Effects of Chondroitinase ABC,Growth Factors and Locomotor Training following Compressive Spinal Cord Injury on Neuroanatomical Plasticity and Kinematics

While several cellular and pharmacological treatments have been evaluated following spinal cord injury (SCI) in animal models, it is increasingly recognized that approaches to address the glial scar, including the use of chondroitinase ABC (ChABC), can facilitate neuroanatomical plasticity. Moreover, increasing evidence suggests that combinatorial strategies are key to unlocking the plasticity that is enabled by ChABC. Given this, we evaluated the anatomical and functional consequences of ChABC in a combinatorial approach that also included growth factor (EGF, FGF2 and PDGF-AA) treatments and daily treadmill training on the recovery of hindlimb locomotion in rats with mid thoracic clip compression SCI. Using quantitative neuroanatomical and kinematic assessments, we demonstrate that the combined therapy significantly enhanced the neuroanatomical plasticity of major descending spinal tracts such as corticospinal and serotonergic-spinal pathways. Additionally, the pharmacological treatment attenuated chronic astrogliosis and inflammation at and adjacent to the lesion with the modest synergistic effects of treadmill training. We also observed a trend for earlier recovery of locomotion accompanied by an improvement of the overall angular excursions in rats treated with ChABC and growth factors in the first 4 weeks after SCI. At the end of the 7-week recovery period, rats from all groups exhibited an impressive spontaneous recovery of the kinematic parameters during locomotion on treadmill. However, although the combinatorial treatment led to clear chronic neuroanatomical plasticity, these structural changes did not translate to an additional long-term improvement of locomotor parameters studied including hindlimb-forelimb coupling. These findings demonstrate the beneficial effects of combined ChABC, growth factors and locomotor training on the plasticity of the injured spinal cord and the potential to induce earlier neurobehavioral recovery. However, additional approaches such as stem cell therapies or a more adapted treadmill training protocol may be required to optimize this repair strategy in order to induce sustained functional locomotor improvement.     

Transplants of Human Mesenchymal Stem Cells Improve Functional Recovery After Spinal Cord Injury in the Rat

Human mesenchymal stem cells (hMSCs) derived from adult bone marrow represent a potentially useful source of cells for cell replacement therapy after nervous tissue damage. They can be expanded in culture and reintroduced into patients as autografts or allografts with unique immunologic properties. The aim of the present study was to investigate (i) survival, migration, differentiation properties of hMSCs transplanted into non-immunosuppressed rats after spinal cord injury (SCI) and (ii) impact of hMSC transplantation on functional recovery. Seven days after SCI, rats received i.v. injection of hMSCs (2×10⁶ in 0.5 mL DMEM) isolated from adult healthy donors. Functional recovery was assessed by Basso–Beattie–Bresnahan (BBB) score weekly for 28 days. Our results showed gradual improvement of locomotor function in transplanted rats with statistically significant differences at 21 and 28 days. Immunocytochemical analysis using human nuclei (NUMA) and BrdU antibodies confirmed survival and migration of hMSCs into the injury site. Transplanted cells were found to infiltrate mainly into the ventrolateral white matter tracts, spreading also to adjacent segments located rostro-caudaly to the injury epicenter. In double-stained preparations, hMSCs were found to differentiate into oligodendrocytes (APC), but not into cells expressing neuronal markers (NeuN). Accumulation of GAP-43 regrowing axons within damaged white matter tracts after transplantation was observed. Our findings indicate that hMSCs may facilitate recovery from spinal cord injury by remyelinating spared white matter tracts and/or by enhancing axonal growth. In addition, low immunogenicity of hMSCs was confirmed by survival of donor cells without immunosuppressive treatment.     

Histological and Functional Benefit Following Transplantation of Motor Neuron Progenitors to the Injured Rat Spinal Cord

Background

Motor neuron loss is characteristic of cervical spinal cord injury (SCI) and contributes to functional deficit.

Methodology/Principal Findings

In order to investigate the amenability of the injured adult spinal cord to motor neuron differentiation, we transplanted spinal cord injured animals with a high purity population of human motor neuron progenitors (hMNP) derived from human embryonic stem cells (hESCs). In vitro, hMNPs displayed characteristic motor neuron-specific markers, a typical electrophysiological profile, functionally innervated human or rodent muscle, and secreted physiologically active growth factors that caused neurite branching and neuronal survival. hMNP transplantation into cervical SCI sites in adult rats resulted in suppression of intracellular signaling pathways associated with SCI pathogenesis, which correlated with greater endogenous neuronal survival and neurite branching. These neurotrophic effects were accompanied by significantly enhanced performance on all parameters of the balance beam task, as compared to controls. Interestingly, hMNP transplantation resulted in survival, differentiation, and site-specific integration of hMNPs distal to the SCI site within ventral horns, but hMNPs near the SCI site reverted to a neuronal progenitor state, suggesting an environmental deficiency for neuronal maturation associated with SCI.

Conclusions/Significance

These findings underscore the barriers imposed on neuronal differentiation of transplanted cells by the gliogenic nature of the injured spinal cord, and the physiological relevance of transplant-derived neurotrophic support to functional recovery.     

Sox11 promotes endogenous neurogenesis and locomotor recovery in mice spinal cord injury

We introduced a lentiviral vector containing the Sox11 gene into injured spinal cords of mice to evaluate the therapeutic potential of Sox11 in spinal cord injury. Sox11 markedly improved locomotor recovery after spinal cord injury and this recovery was accompanied by an up-regulation of Nestin/Doublecortin expression in the injured spinal cord. Sox11 was mainly located in endogenous neural stem cells lining the central canal and in newly-generated neurons in the spinal cord. In addition, Sox 11 significantly induced expressions of BDNF in the spinal cords of LV-Sox11-treated mice. We concluded that Sox11 induced activation of endogenous neural stem cells into neuronal determination and migration within the injured spinal cord. The resultant increase of BDNF at the injured site might form a distinct neurogenic niche which induces a final neuronal differentiation of these neural stem cells. Enhancing Sox11 expression to induce neurogenic differentiation of endogenous neural stem cells after injury may be a promising strategy in restorative therapy after SCI in mammals.     

Polydatin promotes the neuronal differentiation of bone marrow mesenchymal stem cells in vitro and in vivo: Involvement of Nrf2 signalling pathway

Bone marrow mesenchymal stem cell (BMSC) transplantation represents a promising repair strategy following spinal cord injury (SCI), although the therapeutic effects are minimal due to their limited neural differentiation potential. Polydatin (PD), a key component of the Chinese herb Polygonum cuspidatum, exerts significant neuroprotective effects in various central nervous system disorders and protects BMSCs against oxidative injury. However, the effect of PD on the neuronal differentiation of BMSCs, and the underlying mechanisms remain inadequately understood. In this study, we induced neuronal differentiation of BMSCs in the presence of PD, and analysed the Nrf2 signalling and neuronal differentiation markers using routine molecular assays. We also established an in vivo model of SCI and assessed the locomotor function of the mice through hindlimb movements and electrophysiological measurements. Finally, tissue regeneration was evaluated by H&E staining, Nissl staining and transmission electron microscopy. PD (30 μmol/L) markedly facilitated BMSC differentiation into neuron‐like cells by activating the Nrf2 pathway and increased the expression of neuronal markers in the transplanted BMSCs at the injured spinal cord sites. Furthermore, compared with either monotherapy, the combination of PD and BMSC transplantation promoted axonal rehabilitation, attenuated glial scar formation and promoted axonal generation across the glial scar, thereby enhancing recovery of hindlimb locomotor function. Taken together, PD augments the neuronal differentiation of BMSCs via Nrf2 activation and improves functional recovery, indicating a promising new therapeutic approach against SCI.     

Alterations in the expression of the apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE/ref-1) and DNA damage in the caudal region of acute and chronic spinal cord injured rats treated by embryonic neural stem cells

The oxidative mechanisms of injury-induced damage of neurons within the spinal cord are not very well understood. We used a model of T8-T9 spinal cord injury (SCI) in the rat to induce neuronal degeneration. In this spinal cord injury model, unilateral avulsion of the spinal cord causes oxidative stress of neurons. We tested the hypothesis that apurinic/apyrimidinic endonuclease (or redox effector factor-1, APE/Ref-1) regulates this neuronal oxidation mechanism in the spinal cord region caudal to the lesion, and that DNA damage is an early upstream signal. The embryonic neural stem cell therapy significantly decreased DNA-damage levels in both study groups - acutely (followed up to 7 days after SCI), and chronically (followed up to 28 days after SCI) injured animals. Meanwhile, mRNA levels of APE/Ref-1 significantly increased after embryonic neural stem cell therapy in acutely and chronically injured animals when compared to acute and chronic sham groups. Our data has demonstrated that an increase of APE/Ref-1 mRNA levels in the caudal region of spinal cord strongly correlated with DNA damage after traumatic spinal cord injury. We suggest that DNA damage can be observed both in lesional and caudal regions of the acutely and chronically injured groups, but DNA damage is reduced with embryonic neural stem cell therapy.     

Transplantation of D15A-Expressing Glial-Restricted-Precursor-Derived Astrocytes Improves Anatomical and Locomotor Recovery after Spinal Cord Injury

The transplantation of neural stem/progenitor cells is a promising therapeutic strategy for spinal cord injury (SCI). In this study, we tested whether combination of neurotrophic factors and transplantation of glial-restricted precursor (GRPs)-derived astrocytes (GDAs) could decrease the injury and promote functional recovery after SCI. We developed a protocol to quickly produce a sufficiently large, homogenous population of young astrocytes from GRPs, the earliest arising progenitor cell population restricted to the generation of glia. GDAs expressed the axonal regeneration promoting substrates, laminin and fibronectin, but not the inhibitory chondroitin sulfate proteoglycans (CSPGs). Importantly, GDAs or its conditioned medium promoted the neurite outgrowth of dorsal root ganglion neurons in vitro. GDAs were infected with retroviruses expressing EGFP or multi-neurotrophin D15A and transplanted into the contused adult thoracic spinal cord at 8 days post-injury. Eight weeks after transplantation, the grafted GDAs survived and integrated into the injured spinal cord. Grafted GDAs expressed GFAP, suggesting they remained astrocyte lineage in the injured spinal cord. But it did not express CSPG. Robust axonal regeneration along the grafted GDAs was observed. Furthermore, transplantation of D15A-GDAs significantly increased the spared white matter and decreased the injury size compared to other control groups. More importantly, transplantation of D15A-GDAs significantly improved the locomotion function recovery shown by BBB locomotion scores and Tredscan footprint analyses. However, this combinatorial strategy did not enhance the aberrant synaptic connectivity of pain afferents, nor did it exacerbate posttraumatic neuropathic pain. These results demonstrate that transplantation of D15A-expressing GDAs promotes anatomical and locomotion recovery after SCI, suggesting it may be an effective therapeutic approach for SCI.     

Interleukin-17A regulates ependymal cell proliferation and functional recovery after spinal cord injury in mice

Ependymal cells have been suggested to act as neural stem cells and exert beneficial effects after spinal cord injury (SCI). However, the molecular mechanism underlying ependymal cell regulation after SCI remains unknown. To examine the possible effect of IL-17A on ependymal cell proliferation after SCI, we locally administrated IL-17A neutralizing antibody to the injured spinal cord of a contusion SCI mouse model, and revealed that IL-17A neutralization promoted ependymal cell proliferation, which was paralleled by functional recovery and axonal reorganization of both the corticospinal tract and the raphespinal tract. Further, to test whether ependymal cell-specific manipulation of IL-17A signaling is enough to affect the outcomes of SCI, we generated ependymal cell-specific conditional IL-17RA-knockout mice and analyzed their anatomical and functional response to SCI. As a result, conditional knockout of IL-17RA in ependymal cells enhanced both axonal growth and functional recovery, accompanied by an increase in mRNA expression of neurotrophic factors. Thus, Ependymal cells may enhance the regenerative process partially by secreting neurotrophic factors, and IL-17A stimulation negatively regulates this beneficial effect. Molecular manipulation of ependymal cells might be a viable strategy for improving functional recovery.Subject terms: Neuroimmunology, Spinal cord injury     

Inhibition of Epidermal Growth Factor Receptor Improves Myelination and Attenuates Tissue Damage of Spinal Cord Injury

Preventing demyelination and promoting remyelination of denuded axons are promising therapeutic strategies for spinal cord injury (SCI). Epidermal growth factor receptor (EGFR) inhibition was reported to benefit the neural functional recovery and the axon regeneration after SCI. However, its role in de- and remyelination of axons in injured spinal cord is unclear. In the present study, we evaluated the effects of EGFR inhibitor, PD168393 (PD), on the myelination in mouse contusive SCI model. We found that expression of myelin basic protein (MBP) in the injured spinal cords of PD treated mice was remarkably elevated. The density of glial precursor cells and oligodendrocytes (OLs) was increased and the cell apoptosis in lesions was attenuated after PD168393 treatment. Moreover, PD168393 treatment reduced both the numbers of OX42 + microglial cells and glial fibrillary acidic protein + astrocytes in damaged area of spinal cords. We thus conclude that the therapeutic effects of EGFR inhibition after SCI involves facilitating remyelination of the injured spinal cord, increasing of oligodendrocyte precursor cells and OLs, as well as suppressing the activation of astrocytes and microglia/macrophages.     

Sitagliptin improves functional recovery via GLP‐1R‐induced anti‐apoptosis and facilitation of axonal regeneration after spinal cord injury

Axon growth and neuronal apoptosis are considered to be crucial therapeutic targets against spinal cord injury (SCI). Growing evidences have reported stimulation of glucagon‐like peptide‐1 (GLP‐1)/GLP‐1 receptor (GLP‐1R) signalling axis provides neuroprotection in experimental models of neurodegeneration disease. Endogenous GLP‐1 is rapidly degraded by dipeptidyl peptidase‐IV (DPP4), resulting in blocking of GLP‐1/GLP1R signalling process. Sitagliptin, a highly selective inhibitor of DPP4, has approved to have beneficial effects on diseases in which neurons damaged. However, the roles and the underlying mechanisms of sitagliptin in SCI repairing remain unclear. In this study, we used a rat model of SCI and PC12 cells/primary cortical neurons to explore the mechanism of sitagliptin underlying SCI recovery. We discovered the expression of GLP‐1R decreased in the SCI model. Administration of sitagliptin significantly increased GLP‐1R protein level, alleviated neuronal apoptosis, enhanced axon regeneration and improved functional recovery following SCI. Nevertheless, treatment with exendin9‐39, a GLP‐1R inhibitor, remarkably reversed the protective effect of sitagliptin. Additionally, we detected the AMPK/PGC‐1α signalling pathway was activated by sitagliptin stimulating GLP‐1R. Taken together, sitagliptin may be a potential agent for axon regrowth and locomotor functional repair via GLP‐1R‐induced AMPK/ PGC‐1α signalling pathway after SCI.     

Omega-3 fatty acids and neurological injury

Studies with omega-3 polyunsaturated fatty acids (PUFA) have shown that these compounds have therapeutic potential in several indications in neurology and psychiatry. Acute spinal cord injury (SCI) is an event with devastating consequences, and no satisfactory treatment is available at present. The pathogenetic mechanisms associated with SCI include excitotoxicity, increased oxidation and inflammation. We review here our recent studies, which suggest that omega-3 PUFA have significant neuroprotective potential in spinal cord trauma. In a first study, we administered an intravenous bolus of alpha-linolenic acid (LNA) or docosahexaenoic acid (DHA) 30 min after spinal cord hemisection injury in adult rats. The omega-3 PUFA led to increased neuronal and glial survival, and a significantly improved neurological outcome. In subsequent studies, we tested DHA in a more severe compression model of SCI. We also explored a combined acute and chronic treatment regime using DHA. Saline or DHA was administered intravenously 30 min after compression of the spinal cord. After injury, the saline group received a standard control diet, whereas DHA-injected animals received either a control or a DHA-enriched diet for 6 weeks following injury. We assessed locomotor recovery and analysed markers for cell survival and axonal damage, and we also investigated the effects of the treatment on the inflammatory reaction and the oxidative stress that follow SCI. We showed that the acute DHA treatment is neuroprotective after compression SCI, even if the treatment is delayed up to an hour after injury. The DHA injection led to an increased neuronal and glial cell survival, and the effect of the DHA injection was amplified by addition of DHA to the diet. Rats treated with a DHA injection and a DHA-enriched diet performed significantly better at 6 weeks in terms of neurological outcome. The analysis of the tissue after DHA administration showed that the fatty acid significantly reduced lipid peroxidation, protein oxidation and RNA/DNA oxidation, and the induction of COX-2. Parallel studies in a facial nerve injury model in mice also showed pro-regenerative effects of chronic dietary administration of DHA after nerve lesion. These observations suggest that treatment with omega-3 PUFA could represent a promising therapeutic approach in the management of neurological injury.     

The Multifaceted Effects of Agmatine on Functional Recovery after Spinal Cord Injury through Modulations of BMP-2/4/7 Expressions in Neurons and Glial Cells

Presently, few treatments for spinal cord injury (SCI) are available and none have facilitated neural regeneration and/or significant functional improvement. Agmatine (Agm), a guanidinium compound formed from decarboxylation of L-arginine by arginine decarboxylase, is a neurotransmitter/neuromodulator and been reported to exert neuroprotective effects in central nervous system injury models including SCI. The purpose of this study was to demonstrate the multifaceted effects of Agm on functional recovery and remyelinating events following SCI. Compression SCI in mice was produced by placing a 15 g/mm² weight for 1 min at thoracic vertebra (Th) 9 segment. Mice that received an intraperitoneal (i.p.) injection of Agm (100 mg/kg/day) within 1 hour after SCI until 35 days showed improvement in locomotor recovery and bladder function. Emphasis was made on the analysis of remyelination events, neuronal cell preservation and ablation of glial scar area following SCI. Agm treatment significantly inhibited the demyelination events, neuronal loss and glial scar around the lesion site. In light of recent findings that expressions of bone morphogenetic proteins (BMPs) are modulated in the neuronal and glial cell population after SCI, we hypothesized whether Agm could modulate BMP- 2/4/7 expressions in neurons, astrocytes, oligodendrocytes and play key role in promoting the neuronal and glial cell survival in the injured spinal cord. The results from computer assisted stereological toolbox analysis (CAST) demonstrate that Agm treatment dramatically increased BMP- 2/7 expressions in neurons and oligodendrocytes. On the other hand, BMP- 4 expressions were significantly decreased in astrocytes and oligodendrocytes around the lesion site. Together, our results reveal that Agm treatment improved neurological and histological outcomes, induced oligodendrogenesis, protected neurons, and decreased glial scar formation through modulating the BMP- 2/4/7 expressions following SCI.     

Integration and Long Distance Axonal Regeneration in the Central Nervous System from Transplanted Primitive Neural Stem Cells

Spinal cord injury (SCI) results in devastating motor and sensory deficits secondary to disrupted neuronal circuits and poor regenerative potential. Efforts to promote regeneration through cell extrinsic and intrinsic manipulations have met with limited success. Stem cells represent an as yet unrealized therapy in SCI. Recently, we identified novel culture methods to induce and maintain primitive neural stem cells (pNSCs) from human embryonic stem cells. We tested whether transplanted human pNSCs can integrate into the CNS of the developing chick neural tube and injured adult rat spinal cord. Following injection of pNSCs into the developing chick CNS, pNSCs integrated into the dorsal aspects of the neural tube, forming cell clusters that spontaneously differentiated into neurons. Furthermore, following transplantation of pNSCs into the lesioned rat spinal cord, grafted pNSCs survived, differentiated into neurons, and extended long distance axons through the scar tissue at the graft-host interface and into the host spinal cord to form terminal-like structures near host spinal neurons. Together, these findings suggest that pNSCs derived from human embryonic stem cells differentiate into neuronal cell types with the potential to extend axons that associate with circuits of the CNS and, more importantly, provide new insights into CNS integration and axonal regeneration, offering hope for repair in SCI.