Effects of Wnt5a overexpression in spinal cord injury

Accordingly to its known function in corticospinal tract (CST) developmental growth, previous reports have shown an inhibitory role of Wnt5a in CST regeneration after spinal cord injury (SCI). Interestingly, it has been subsequently demonstrated that Wnt5a also modulates the developmental growth of non-CST axons and that different Wnt5a receptors are expressed in neurons, oligodendrocytes, NG2+ glial precursors and reactive microglia/macrophages and astrocytes after SCI. However, the role of Wnt5a in the response of these cell types, in the regeneration of non-CST axons and in functional recovery after SCI is currently unknown. To evaluate this, rats were subjected to spinal cord contusion and injected with a lentiviral vector generated to overexpress Wnt5a. Histological analyses were performed in spinal cord sections processed for the visualization of myelin, oligodendrocytes, neurons, microglia/macrophages, astrocytes, NG2+ glial precursors and serotonergic axons. Motor and bladder function recovery were also assessed. Further advancing our knowledge on the role of Wnt5a in SCI, we found that, besides its previously reported functions, Wnt5a overexpression elicits a reduction on neuronal cell density, the accumulation of NG2+ glial precursors and the descending serotonergic innervation in the affected areas, along with impairment of motor and bladder function recovery after SCI.     

Aquaporin-4 Mitigates Retrograde Degeneration of Rubrospinal Neurons by Facilitating Edema Clearance and Glial Scar Formation After Spinal Cord Injury in Mice

Atrophy of upper motor neurons hampers axonal regeneration and functional recovery following spinal cord injury (SCI). Apart from the severity of primary injury, a series of secondary pathological damages including spinal cord edema and glial scar formation affect the fate of injured upper motor neurons. The aquaporin-4 (AQP4) water channel plays a critical role in water homeostasis and migration of astrocytes in the central nervous system, probably offering a new therapeutic target for protecting against upper motor neuron degeneration after SCI. To test this hypothesis, we examined the effect of AQP4 deficiency on atrophy of rubrospinal neurons after unilateral rubrospinal tract transection at the fourth cervical level in mice. AQP4 gene knockout (AQP4^?/?) mice exhibited high extent of spinal cord edema at 72 h after lesion compared with wild-type littermates. AQP4^?/? mice showed impairments in astrocyte migration toward the transected site with a greater lesion volume at 1 week after surgery and glial scar formation with a larger cyst volume at 6 weeks. More severe atrophy and loss of axotomized rubrospinal neurons as well as axonal degeneration in the rubrospinal tract rostral to the lesion were observed in AQP4^?/? mice at 6 weeks after SCI. AQP4 expression was downregulated at the lesioned spinal segment at 3 days and 1 week after injury, but upregulated at 6 weeks. These results demonstrated that AQP4 not only mitigates spinal cord damage but also ameliorates retrograde degeneration of rubrospinal neurons by promoting edema clearance and glial scar formation after laceration SCI. This finding supports the notion that AQP4 may be a promising therapeutic target for SCI.     

Role of Cell Cycle Proteins in CNS Injury

Following trauma or ischemia to the central nervous system (CNS), there is a marked increase in the expression of cell cycle-related proteins. This up-regulation is associated with apoptosis of post-mitotic cells, including neurons and oligodendrocytes, both in vitro and in vivo. Cell cycle activation also induces proliferation of astrocytes and microglia, contributing to the glial scar and microglial activation with release of inflammatory factors. Treatment with cell cycle inhibitors in CNS injury models inhibits glial scar formation and neuronal cell death, resulting in substantially decreased lesion volumes and improved behavioral recovery. Here we critically review the role of cell cycle pathways in the pathophysiology of experimental stroke, traumatic brain injury and spinal cord injury, and discuss the potential of cell cycle inhibitors as neuroprotective agents. Special issue dedicated to Dr. Moussa Youdim.     

More attention on glial cells to have better recovery after spinal cord injury

Functional improvement after spinal cord injury remains an unsolved difficulty. Glial scars, a major component of SCI lesions, are very effective in improving the rate of this recovery. Such scars are a result of complex interaction mechanisms involving three major cells, namely, astrocytes, oligodendrocytes, and microglia. In recent years, scientists have identified two subtypes of reactive astrocytes, namely, A1 astrocytes that induce the rapid death of neurons and oligodendrocytes, and A2 astrocytes that promote neuronal survival. Moreover, recent studies have suggested that the macrophage polarization state is more of a continuum between M1 and M2 macrophages. M1 macrophages that encourage the inflammation process kill their surrounding cells and inhibit cellular proliferation. In contrast, M2 macrophages promote cell proliferation, tissue growth, and regeneration. Furthermore, the ability of oligodendrocyte precursor cells to differentiate into adult oligodendrocytes or even neurons has been reviewed. Here, we first scrutinize recent findings on glial cell subtypes and their beneficial or detrimental effects after spinal cord injury. Second, we discuss how we may be able to help the functional recovery process after injury.     

Reactive Astrocytes in Glial Scar Attract Olfactory Ensheathing Cells Migration by Secreted TNF-α in Spinal Cord Lesion of Rat

Background

After spinal cord injury (SCI), the formation of glial scar contributes to the failure of injured adult axons to regenerate past the lesion. Increasing evidence indicates that olfactory ensheathing cells (OECs) implanted into spinal cord are found to migrate into the lesion site and induce axons regeneration beyond glial scar and resumption of functions. However, little is known about the mechanisms of OECs migrating from injection site to glial scar/lesion site.

Methods and Findings

In the present study, we identified a link between OECs migration and reactive astrocytes in glial scar that was mediated by the tumor necrosis factor-α (TNF-α). Initially, the Boyden chamber migration assay showed that both glial scar tissue and reactive astrocyte-conditioned medium promoted OECs migration in vitro. Reactive astrocyte-derived TNF-α and its type 1 receptor TNFR1 expressed on OECs were identified to be responsible for the promoting effect on OECs migration. TNF-α-induced OECs migration was demonstrated depending on activation of the extracellular signal-regulated kinase (ERK) signaling cascades. Furthermore, TNF-α secreted by reactive astrocytes in glial scar was also showed to attract OECs migration in a spinal cord hemisection injury model of rat.

Conclusions

These findings showed that TNF-α was released by reactive astrocytes in glial scar and attracted OECs migration by interacting with TNFR1 expressed on OECs via regulation of ERK signaling. This migration-attracting effect of reactive astrocytes on OECs may suggest a mechanism for guiding OECs migration into glial scar, which is crucial for OECs-mediated axons regrowth beyond the spinal cord lesion site.     

Inhibition of NADPH Oxidase Activation in Oligodendrocytes Reduces Cytotoxicity Following Trauma

Spinal cord injury is a debilitating neurological disorder that initiates a cascade of cellular events that result in a period of secondary damage that can last for months after the initial trauma. The ensuing outcome of these prolonged cellular perturbations is the induction of neuronal and glial cell death through excitotoxic mechanisms and subsequent free radical production. We have previously shown that astrocytes can directly induce oligodendrocyte death following trauma, but the mechanisms regulating this process within the oligodendrocyte remain unclear. Here we provide evidence demonstrating that astrocytes directly regulate oligodendrocyte death after trauma by inducing activation of NADPH oxidase within oligodendrocytes. Spinal cord injury resulted in a significant increase in oxidative damage which correlated with elevated expression of the gp91 phox subunit of the NADPH oxidase enzyme. Immunohistochemical analysis confirmed the presence of gp91 phox in oligodendrocytes in vitro and at 1 week following spinal cord injury. Exposure of oligodendrocytes to media from injured astrocytes resulted in an increase in oligodendrocyte NADPH oxidase activity. Inhibition of NADPH oxidase activation was sufficient to attenuate oligodendrocyte death in vitro and at 1 week following spinal cord injury, suggesting that excitotoxicity of oligodendrocytes after trauma is dependent on the intrinsic activation of the NADPH oxidase enzyme. Acute administration of the NADPH oxidase inhibitor apocynin and the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate channel blocker 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione significantly improved locomotor behavior and preserved descending axon fibers following spinal cord injury. These studies lead to a better understanding of oligodendrocyte death after trauma and identify potential therapeutic targets in disorders involving demyelination and oligodendrocyte death.     

Chondroitinase and growth factors enhance activation and oligodendrocyte differentiation of endogenous neural precursor cells after spinal cord injury

The adult spinal cord harbours a population of multipotent neural precursor cells (NPCs) with the ability to replace oligodendrocytes. However, despite this capacity, proliferation and endogenous remyelination is severely limited after spinal cord injury (SCI). In the post-traumatic microenvironment following SCI, endogenous spinal NPCs mainly differentiate into astrocytes which could contribute to astrogliosis that exacerbate the outcomes of SCI. These findings emphasize a key role for the post-SCI niche in modulating the behaviour of spinal NPCs after SCI. We recently reported that chondroitin sulphate proteoglycans (CSPGs) in the glial scar restrict the outcomes of NPC transplantation in SCI by reducing the survival, migration and integration of engrafted NPCs within the injured spinal cord. These inhibitory effects were attenuated by administration of chondroitinase (ChABC) prior to NPC transplantation. Here, in a rat model of compressive SCI, we show that perturbing CSPGs by ChABC in combination with sustained infusion of growth factors (EGF, bFGF and PDGF-AA) optimize the activation and oligodendroglial differentiation of spinal NPCs after injury. Four days following SCI, we intrathecally delivered ChABC and/or GFs for seven days. We performed BrdU incorporation to label proliferating cells during the treatment period after SCI. This strategy increased the proliferation of spinal NPCs, reduced the generation of new astrocytes and promoted their differentiation along an oligodendroglial lineage, a prerequisite for remyelination. Furthermore, ChABC and GF treatments enhanced the response of non-neural cells by increasing the generation of new vascular endothelial cells and decreasing the number of proliferating macrophages/microglia after SCI. In conclusions, our data strongly suggest that optimization of the behaviour of endogenous spinal NPCs after SCI is critical not only to promote endogenous oligodendrocyte replacement, but also to reverse the otherwise detrimental effects of their activation into astrocytes which could negatively influence the repair process after 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.     

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.     

Neural progenitors isolated from newborn rat spinal cords differentiate into neurons and astroglia

Permanent functional deficit in patients with spinal cord injury (SCI) is in part due to severe neural cell death. Therefore, cell replacement using stem cells and neural progenitors that give rise to neurons and glia is thought to be a potent strategy to promote tissue repair after SCI. Many studies have shown that stem cells and neural progenitors can be isolated from embryonic, postnatal and adult spinal cords. Recently, we isolated neural progenitors from newborn rat spinal cords. In general, the neural progenitors grew as spheres in culture, and showed immunoreactivity to a neural progenitor cellular marker, nestin. They were found to proliferate and differentiate into glial fibrillary acidic protein-positive astroglia and multiple neuronal populations, including GABAergic and cholinergic neurons. Neurotrophin 3 and neurotrophin 4 enhanced the differentiation of neural progenitors into neurons. Furthermore, the neural progenitors that were transplanted into contusive spinal cords were found to survive and have migrated in the spinal cord rostrally and caudally over 8 mm to the lesion center 7 days after injury. Thus, the neural progenitors isolated from newborn rat spinal cords in combination with neurotrophic factors may provide a tool for cell therapy in SCI patients.     

Mixed primary culture and clonal analysis provide evidence that NG2 proteoglycan-expressing cells after spinal cord injury are glial progenitors

NG2(+) cells in the adult rat spinal cord proliferate after spinal cord injury (SCI) and are postulated to differentiate into mature glia to replace some of those lost to injury. To further study these putative endogenous precursors, tissue at 3 days after SCI or from uninjured adults was dissociated, myelin partially removed and replicate cultures grown in serum-containing or serum-free medium with or without growth factors for up to 7 days in vitro (DIV). Cell yield after SCI was 5-6 times higher than from the normal adult. Most cells were OX42(+) microglia/macrophages but there were also more than twice the normal number of NG2(+) cells. Most of these coexpressed A2B5 or nestin, as would be expected for glial progenitors. Few cells initially expressed mature astrocyte (GFAP) or oligodendrocyte (CC1) markers, but more did at 7 DIV, suggesting differentiation of glial precursors in vitro. To test the hypothesis that NG2(+) cells after SCI express progenitor-like properties, we prepared free-floating sphere and single cell cultures from purified suspension of NG2(+) cells from injured spinal cord. We found that sphere cultures could be passaged in free-floating subcultures, and upon attachment the spheres clonally derived from an acutely purified single cell differentiated into oligodendrocytes and rarely astrocytes. Taken together, these data support the hypothesis that SCI stimulates proliferation of NG2(+) cells that are glial progenitor cells. Better understanding the intrinsic properties of the NG2(+) cells stimulated by SCI may permit future therapeutic manipulations to improve recovery after SCI.     

Neural stem/progenitor cells from the adult human spinal cord are multipotent and self-renewing and differentiate after transplantation

Neural stem/progenitor cell (NSPC) transplantation is a promising therapy for spinal cord injury (SCI). However, little is known about NSPC from the adult human spinal cord as a donor source. We demonstrate for the first time that multipotent and self-renewing NSPC can be cultured, passaged and transplanted from the adult human spinal cord of organ transplant donors. Adult human spinal cord NSPC require an adherent substrate for selection and expansion in EGF (epidermal growth factor) and FGF2 (fibroblast growth factor) enriched medium. NSPC as an adherent monolayer can be passaged for at least 9 months and form neurospheres when plated in suspension culture. In EGF/FGF2 culture, NSPC proliferate and primarily express nestin and Sox2, and low levels of markers for differentiating cells. Leukemia inhibitory factor (LIF) promotes NSPC proliferation and significantly enhances GFAP expression in hypoxia. In differentiating conditions in the presence of serum, these NSPC show multipotentiality, expressing markers of neurons, astrocytes, and oligodendrocytes. Dibutyryl cyclic AMP (dbcAMP) significantly enhances neuronal differentiation. We transplanted the multipotent NSPC into SCI rats and show that the xenografts survive, are post-mitotic, and retain the capacity to differentiate into neurons and glia.Together, these findings reveal that multipotent self-renewing NSPC cultured and passaged from adult human spinal cords of organ transplant donors, respond to exogenous factors that promote selective differentiation, and survive and differentiate after transplantation into the injured spinal cord.     

X-irradiation reduces lesion scarring at the contusion site of adult rat spinal cord

Spinal cord injury (SCI) results in cell death and tissue destruction, and ultimately cavitation followed by the formation of lesion scars at the injury site. The lesion scars include an astrocytic component (glial scar) and a fibroblastic component (connective tissue scar). The purpose of the present study is to determine if X-irradiation could minimize the formation of lesion scars and reduce the levels of chondroitin sulfate proteoglycans (CSPGs) in the contusion SCI model of the adult rat. Two weeks after SCI, a connective tissue scar formed at the injury site consisting primarily of fibroblasts and exhibits strong CSPG immunoreactivity. The fibroblasts might originate from the connective tissue of pia mater or arachnoid mater. At the same time, reactive astrocytes in the spared tissue accumulate surrounding the lesion cavity to form a thick glial scar with significant enhancement of glial fibrillary acidic protein (GFAP) and CSPG immunoreactivity. After X-irradiation (40 Gy) of the injury site 2 days post-injury, that results in an attenuated dose to the lesion, the connective tissue scar was not observed, and accordingly, almost no CSPG immunoreactivity was detected at this area. Meanwhile, the glial scar and its CSPG immunoreactivity were prominently reduced. X-irradiation did not show significant improvement in locomotor recovery, but resulted in a slight delay of body weight recovery following injury. This preparative treatment could be used to reduce secondary scarring in the lesion resulting in an enriched site for further treatment such as growth related transplantation.     

In vivo analysis of glial cell phenotypes during a viral demyelinating disease in mice

C57 BL/6N mice injected intracranially with the A59 strain of mouse hepatitis virus exhibit extensive viral replication in glial cells of the spinal cord and develop demyelinating lesions followed by virus clearing and remyelination. To study how different glial cell types are affected by the disease process, we combine three-color immunofluorescence labeling with tritiated thymidine autoradiography on 1-micron frozen sections of spinal cord. We use three different glial cell specific antibodies (a) to 2',3' cyclic-nucleotide 3' phosphohydrolase (CNP) expressed by oligodendrocytes, (b) to glial fibrillary acidic protein (GFAP) expressed by astrocytes, and (c) the O4 antibody which binds to O-2A progenitor cells in the rat. These progenitor cells, which give rise to oligodendrocytes and type 2 astrocytes and react with the O4 antibody in the adult central nervous system, were present but rare in the spinal cord of uninfected mice. In contrast, cells with the O-2A progenitor phenotype (O4 + only) were increased in number at one week post viral inoculation (1 WPI) and were the only immunostained cells labeled at that time by a 2-h in vivo pulse of tritiated thymidine. Both GFAP+ only and GFAP+, O4+ astrocytes were also increased in the spinal cord at 1 WPI. Between two and four WPI, the infected spinal cord was characterized by the loss of (CNP+, O4+) oligodendrocytes within demyelinating lesions and the presence of O-2A progenitor cells and O4+, GFAP+ astrocytes, both of which could be labeled with thymidine. As remyelination proceeded, CNP immunostaining returned to near normal and tritiated thymidine injected previously during the demyelinating phase now appeared in CNP+ oligodendrocytes. Thus O4 positive O-2A progenitor cells proliferate early in the course of the demyelinating disease, while CNP positive oligodendrocytes do not. The timing of events suggests that the O-2A progenitors may give rise to new oligodendrocytes and to type 2 astrocytes, both of which are likely to be instrumental in the remyelination process.     

Lentiviral vector delivery of short hairpin RNA to NG2 and neurotrophin-3 promotes locomotor recovery in injured rat spinal cord

Background aimsIn this study we investigated the effect of neurotrophin-3 (NT-3) and knockdown of NG2, one of the main inhibitory chondroitin sulfate proteoglycans (CSPG), in the glial scar following spinal cord injury (SCI).MethodsShort hairpin (sh) RNA were designed to target NG2 and were cloned into a lentiviral vector (LV). A LV was also constructed containing NT-3. LV expressing NT-3, shRNA to NG2 or combinations of both vectors were injected directly into contused rat spinal cords 1 week post-injury. Six weeks post-injection of LV, spinal cords were examined by histology for changes in scar size and by immunohistochemistry for changes in expression of CSPG, NT-3, astrocytes, neurons and microglia/macrophages. Motor function was assessed using the Basso, Beattie and Bresnahan (BBB) locomotor scale.ResultsAnimals that received the combination treatment of LV shNG2 and LV NT-3 showed reduced scar size. These animals also showed an increase in levels of neurons and NG2, a decrease in levels of astrocytes and a significant functional recovery as assessed using the BBB locomotor scale at 2 weeks post-treatment.ConclusionsThe improvement in locomotor recovery and decrease in scar size shows the potential of this gene therapy approach as a therapeutic treatment for 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.     

BMP inhibition enhances axonal growth and functional recovery after spinal cord injury

Bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor-β superfamily. BMPs regulate several crucial aspects of embryonic development and organogenesis. The reemergence of BMPs in the injured adult CNS suggests their involvement in the pathogenesis of the lesion. Here, we demonstrate that BMPs are potent inhibitors of axonal regeneration in the adult spinal cord. The expression of BMP-2/4 is elevated in oligodendrocytes and astrocytes around the injury site following spinal cord contusion. Intrathecal administration of noggin – a soluble BMP antagonist—leads to enhanced locomotor activity and reveals significant regrowth of the corticospinal tract after spinal cord contusion. Thus, BMPs play a role in inhibiting axonal regeneration and limiting functional recovery following injury to the CNS.