Triptolide promotes spinal cord repair by inhibiting astrogliosis and inflammation

Spinal cord injury (SCI) is a cause of major neurological disability, and no satisfactory treatment is currently available. Traumatic SCI directly damages the cell bodies and/or processes of neurons and triggers a series of endogenous processes, including neuroinflammatory response and reactive astrogliosis. In this study, we found that triptolide, one of the major active components of the traditional Chinese herb Tripterygium wilfordii Hook F, inhibited astrogliosis and inflammation and promoted spinal cord repair. Triptolide was shown to prevent astrocytes from reactive activation by blocking the JAK2/STAT3 pathway in vitro and in vivo. Furthermore, astrocytic gliosis and glial scar were greatly reduced in injured spinal cord treated with triptolide. Triptolide treatment was also shown to decrease the ED‐1 or CD11b‐positive inflammatory cells at the lesion site. Using neurofilament staining and anterograde tracing, a significantly greater number of regenerative axons were observed in the triptolide‐treated rats. Importantly, behavioral tests revealed that injured rats receiving triptolide had improved functional recovery as assessed by the Basso, Beattie, and Bresnahan open‐field scoring, grid‐walk, and foot‐print analysis. These results suggested that triptolide promoted axon regeneration and locomotor recovery by attenuating glial scaring and inflammation, and shed light on the potential therapeutic benefit for SCI. © 2010 Wiley‐Liss, Inc.     

MicroRNA‐31 is required for astrocyte specification

Previously, we determined microRNA‐31 (miR‐31) is a noncoding tumor suppressive gene frequently deleted in glioblastoma (GBM); miR‐31 suppresses tumor growth, in part, by limiting the activity of NF‐κB. Herein, we expand our previous studies by characterizing the role of miR‐31 during neural precursor cell (NPC) to astrocyte differentiation. We demonstrate that miR‐31 expression and activity is suppressed in NPCs by stem cell factors such as Lin28, c‐Myc, SOX2 and Oct4. However, during astrocytogenesis, miR‐31 is induced by STAT3 and SMAD1/5/8, which mediate astrocyte differentiation. We determined miR‐31 is required for terminal astrocyte differentiation, and that the loss of miR‐31 impairs this process and/or prevents astrocyte maturation. We demonstrate that miR‐31 promotes astrocyte development, in part, by reducing the levels of Lin28, a stem cell factor implicated in NPC renewal. These data suggest that miR‐31 deletions may disrupt astrocyte development and/or homeostasis.     

Blockade of interleukin-6 receptor suppresses reactive astrogliosis and ameliorates functional recovery in experimental spinal cord injury

Endogenous neural stem/progenitor cells (NSPCs) have recently been shown to differentiate exclusively into astrocytes, the cells that are involved in glial scar formation after spinal cord injury (SCI). The microenvironment of the spinal cord, especially the inflammatory cytokines that dramatically increase in the acute phase at the injury site, is considered to be an important cause of inhibitory mechanism of neuronal differentiation following SCI. Interleukin-6 (IL-6), which has been demonstrated to induce NSPCs to undergo astrocytic differentiation selectively through the JAK/STAT pathway in vitro, has also been demonstrated to play a critical role as a proinflammatory cytokine and to be associated with secondary tissue damage in SCI. In this study, we assessed the efficacy of rat anti-mouse IL-6 receptor monoclonal antibody (MR16-1) in the treatment of acute SCI in mice. Immediately after contusive SCI with a modified NYU impactor, mice were intraperitoneally injected with a single dose of MR16-1 (100 microg/g body weight), the lesions were assessed histologically, and the functional recovery was evaluated. MR16-1 not only suppressed the astrocytic diffentiation-promoting effect of IL-6 signaling in vitro but inhibited the development of astrogliosis after SCI in vivo. MR16-1 also decreased the number of invading inflammatory cells and the severity of connective tissue scar formation. In addition, we observed significant functional recovery in the mice treated with MR16-1 compared with control mice. These findings suggest that neutralization of IL-6 signaling in the acute phase of SCI represents an attractive option for the treatment of SCI.     

The adult spinal cord harbors a population of GFAP‐positive progenitors with limited self‐renewal potential

Adult neural stem cells (aNSCs) of the forebrain are GFAP‐expressing cells that are intercalated within ependymal cells of the subventricular zone (SVZ). Cells showing NSCs characteristics in vitro can also be isolated from the periaqueductal region in the adult spinal cord (SC), but contradicting results exist concerning their glial versus ependymal identity. We used an inducible transgenic mouse line (hGFAP‐CreERT2) to conditionally label GFAP‐expressing cells in the adult SVZ and SC periaqueduct, and directly and systematically compared their self‐renewal and multipotential properties in vitro. We demonstrate that a population of GFAP⁺ cells that share the morphology and the antigenic properties of SVZ‐NSCs mostly reside in the dorsal aspect of the central canal (CC) throughout the spinal cord. These cells are non‐proliferative in the intact spinal cord, but incorporate the S‐phase marker EdU following spinal cord injury. Multipotent, clonal YFP‐expressing neurospheres (i.e., deriving from recombined GFAP‐expressing cells) were successfully obtained from both the intact and injured spinal cord. These spheres however showed limited self‐renewal properties when compared with SVZ‐neurospheres, even after spinal cord injury. Altogether, these results demonstrate that significant differences exist in NSCs lineages between neurogenic and non‐neurogenic regions of the adult CNS. Thus, although we confirm that a population of multipotent GFAP⁺ cells co‐exists alongside with multipotent ependymal cells within the adult SC, we identify these cells as multipotent progenitors showing limited self‐renewal properties. GLIA 2013;61:2100–2113     

Contribution of bone marrow‐derived endothelial progenitor cells to neovascularization and astrogliosis following spinal cord injury

Spinal cord injury causes initial mechanical damage, followed by ischemia‐induced, secondary degeneration, worsening the tissue damage. Although endothelial progenitor cells (EPCs) have been reported to play an important role for pathophysiological neovascularization in various ischemic tissues, the EPC kinetics following spinal cord injury have never been elucidated. In this study, we therefore assessed the in vivo kinetics of bone marrow‐derived EPCs by EPC colony‐forming assay and bone marrow transplantation from Tie2/lacZ transgenic mice into wild‐type mice with spinal cord injury. The number of circulating mononuclear cells and EPC colonies formed by the mononuclear cells peaked at day 3 postspinal cord injury. Bone marrow transplantation study revealed that bone marrow‐derived EPCs recruited into the injured spinal cord markedly increased at day 7, when neovascularization and astrogliosis drastically occurred in parallel with axon growth in the damaged tissue. To elucidate further the contribution of EPCs to recovery after spinal cord injury, exogenous EPCs were systemically infused immediately after the injury. The administered EPCs were incorporated into the injured spinal cord and accelerated neovascularization and astrogliosis. These findings suggest that bone marrow‐derived EPCs may contribute to the tissue repair by augmenting neovascularization and astrogliosis following spinal cord injury. © 2012 Wiley Periodicals, Inc.     

Depletion of SASH1, an astrocyte differentiation‐related gene,contributes to functional recovery in spinal cord injury

AimsThis study aimed to evaluate the effects of the depletion of SAM and SH3 domain‐containing protein 1 (SASH1) on functional recovery after spinal cord injury (SCI) and to investigate the possible mechanism of SASH1 knockdown in astrocytes facilitating axonal growth.MethodsSCI model was established in adult rats. SASH1 small interfering RNA (siSASH1) was used to investigate its function. Hindlimb motor function was evaluated by the Basso‐Bresnahan‐Beattie (BBB) assay. The gene expressions were evaluated by the methods of qRT‐PCR, Western‐blotting, ELISA, and immunohistochemistry.ResultsSASH1 knockdown improved the BBB scores after SCI and significantly reduced GFAP expression. In cultured spinal astrocytes, siSASH1 treatment decreased interferon‐γ release and increased brain‐derived neurotrophic factor (BDNF) release. When cocultured with SASH1‐knockdown astrocytes, axonal growth increased. The neuronal tropomyosin receptor kinase B (BDNF receptor) expression increased, especially in the axonal tips. SASH1 expression increased while NSCs differentiated into glial cells, instead of neurons. After SASH1 depletion, differentiated NSCs maintained a higher level of Nestin protein and an increase in BDNF release.ConclusionsThese results indicate that SASH1 acts as an astrocytic differentiation‐maintaining protein, and SASH1 downregulation limits glial activation and contributes toward functional recovery after SCI.     

Expression and detrimental role of hematopoietic prostaglandin D synthase in spinal cord contusion injury

Prostaglandin D(2) (PGD(2) ) is a potent inflammatory mediator, which is implicated in both the initiation and resolution of inflammation in peripheral non-neural tissues. Its role in the central nervous system has not been fully elucidated. Spinal cord injury (SCI) is associated with an acute inflammatory response, which contributes to secondary tissue damage that worsens functional loss. We show here, with the use of hematopoietic prostaglandin D synthase (HPGDS) deficient mice and a HPGDS selective inhibitor (HQL-79), that PGD(2) plays a detrimental role after SCI. We also show that HPGDS is expressed in macrophages in the injured mouse spinal cord and contributes to the increase in PGD(2) in the contused spinal cord. HPGDS(-/-) mice also show reduced secondary tissue damage and reduced expression of the proinflammatory chemokine CXCL10 as well as an increase in IL-6 and TGFβ-1 expression in the injured spinal cord. This was accompanied by a reduction in the expression of the microglia/macrophage activation marker Mac-2 and an increase in the antioxidant metallothionein III. Importantly, HPGDS deficient mice exhibit significantly better locomotor recovery after spinal cord contusion injury than wild-type (Wt) mice. In addition, systemically administered HPGDS inhibitor (HQL-79) also enhanced locomotor recovery after SCI in Wt mice. These data suggest that PGD(2) generated via HPGDS has detrimental effects after SCI and that blocking the activity of this enzyme can be beneficial.     

RIG‐I mediates nonsegmented negative‐sense RNA virus‐induced inflammatory immune responses of primary human astrocytes

While astrocytes produce key inflammatory mediators following exposure to neurotropic nonsegmented negative‐sense RNA viruses such as rabies virus and measles virus, the mechanisms by which resident central nervous system (CNS) cells perceive such viral challenges have not been defined. Recently, several cytosolic DExD/H box RNA helicases including retinoic acid‐inducible gene I (RIG‐I) have been described that function as intracellular sensors of replicative RNA viruses. Here, we demonstrate that primary human astrocytes constitutively express RIG‐I and show that such expression is elevated following exposure to a model neurotropic RNA virus, vesicular stomatitis virus (VSV). Evidence for the functional nature of RIG‐I expression in these cells comes from the observation that this molecule associates with its downstream effector molecule, interferon promoter stimulator‐1, following VSV infection and from the finding that a specific ligand for RIG‐I elicits astrocyte immune responses. Importantly, RIG‐I knockdown significantly reduces inflammatory cytokine production by VSV‐infected astrocytes and inhibits the production of soluble neurotoxic mediators by these cells. These findings directly implicate RIG‐I in the initiation of inflammatory immune responses by human glial cells and provide a potential mechanism underlying the neuronal cell death associated with acute viral CNS infections. © 2010 Wiley‐Liss, Inc.     

Astrogliosis involves activation of retinoic acid-inducible gene-like signaling in the innate immune response after spinal cord injury

Spinal cord injury (SCI) induces a glial response in which astrocytes become activated and produce inflammatory mediators. The molecular basis for regulation of glial-innate immune responses remains poorly understood. Here, we examined the activation of retinoic acid-inducible gene (RIG)-like receptors (RLRs) and their involvement in regulating inflammation after SCI. We show that astrocytes express two intracellular RLRs: RIG-I and melanoma differentiation-associated gene 5. SCI and stretch injury of cultured astrocytes stimulated RLR signaling as determined by phosphorylation of interferon regulatory factor 3 (IRF3) leading to production of type I interferons (IFNs). RLR signaling stimulation with synthetic ribonucleic acid resulted in RLR activation, phosphorylation of IRF3, and increased expression of glial fibrillary acidic protein (GFAP) and vimentin, two hallmarks of reactive astrocytes. Moreover, mitochondrial E3 ubiquitin protein ligase 1, an RLR inhibitor, decreased production of GFAP and vimentin after RIG-I signaling stimulation. Our findings identify a role for RLR signaling and type I IFN in regulating astrocyte innate immune responses after SCI.     

Neurorehabilitation of Upper Extremities in Humans with Sensory‐Motor Impairment

Today most clinical investigators agree that the common denominator for successful therapy in subjects after central nervous system (CNS) lesions is to induce concentrated, repetitive practice of the more affected limb as soon as possible after the onset of impairment. This paper reviews representative methods of neurorehabilitation such as constraining the less affected arm and using a robot to facilitate movement of the affected arm, and focuses on functional electrotherapy promoting the movement recovery. The functional electrical therapy (FET) encompasses three elements: 1) control of movements that are compromised because of the impairment, 2) enhanced exercise of paralyzed extremities, and 3) augmented activity of afferent neural pathway. Liberson et al. 1 first reported an important result of the FET; they applied a peroneal stimulator to enhance functionally essential ankle dorsiflexion during the swing phase of walking. Merletti et al. 2 described a similar electrotherapeutic effect for upper extremities; they applied a two‐channel electronic stimulator and surface electrodes to augment elbow extension and finger extension during different reach and grasp activities. Both electrotherapies resulted in immediate and carry‐over effects caused by systematic application of FET. In studies with subjects after a spinal cord lesion at the cervical level (chronic tetraplegia) 3 - 5 or stroke 6 , it was shown that FET improves grasping and reaching by using the following outcome measures: the Upper Extremity Function Test (UEFT), coordination between elbow and shoulder movement, and the Functional Independence Measure (FIM). Externally applied electrical stimuli provided a strong central sensory input which could be responsible for the changes in the organization of impaired sensory‐motor mechanisms. FET resulted in stronger muscles that were stimulated directly, as well as exercising other muscles. The ability to move paralyzed extremities also provided awareness (proprioception and visual feedback) of enhanced functional ability as being very beneficial for the recovery. FET contributed to the increased range of movement in the affected joints, increased speed of joint rotations, reduced spasticity, and improved functioning measured by the UEFT, the FIM and the Quadriplegia Index of Function (QIF).     

Evidence for the role of PI(3) -kinase-AKT-eNOS signalling pathway in secondary inflammatory process after spinal cord compression injury in mice

Endothelial nitric oxide synthase (eNOS) is a dynamic enzyme tightly controlled by co‐ and post‐translational lipid modifications, phosphorylation and regulated by protein–protein interactions. In this study we have pharmacologically modulated the activation of eNOS, at different post‐translational levels, to assess the role of eNOS‐derived NO and regulatory mechanisms in tissue damage associated with spinal cord injury (SCI). SC trauma was induced by the application of vascular clips (force of 24 g) to the dura via a four‐level T5–T8 laminectomy. SCI in mice resulted in severe trauma characterized by oedema, neutrophil infiltration, and production of inflammatory mediators, tissue damage and apoptosis. LY294002, an inhibitor of phosphatidylinositol 3‐kinase that initiates Akt‐catalysed phosphorylation of eNOS on Ser1179, was administered 1 h before the induction of SCI; 24 h after SCI sections were taken for histological examination and for biochemical studies. In this study we clearly demonstrated that pre‐treatment with LY294002 reversed the increased activation of eNOS and Akt observed following SCI, and developed a severe trauma characterized by oedema, tissue damage and apoptosis (measured by TUNEL staining, Bax, Bcl‐2 and Fas‐L expression). Histological damage also correlated with neutrophil infiltration, assessed as myeloperoxidase activity. Overall these results suggest that activation of the Akt pathway in SC tissue subject to SCI is a protective event, triggered in order to protect the injured tissue through a fine tuning of the endothelial NO pathway.     

A Parkinson's disease gene,DJ‐1, repairs brain injury through Sox9 stabilization and astrogliosis

Defects in repair of damaged brain accumulate injury and contribute to slow‐developing neurodegeneration. Here, we report that a deficiency of DJ‐1, a Parkinson's disease (PD) gene, delays repair of brain injury due to destabilization of Sox9, a positive regulator of astrogliosis. Stereotaxic injection of ATP into the brain striatum produces similar size of acute injury in wild‐type and DJ‐1‐knockout (KO) mice. However, recovery of the injury is delayed in KO mice, which is confirmed by 9.4T magnetic resonance imaging and tyrosine hydroxylase immunostaining. DJ‐1 regulates neurite outgrowth from damaged neurons in a non‐cell autonomous manner. In DJ‐1 KO brains and astrocytes, Sox9 protein levels are decreased due to enhanced ubiquitination, resulting in defects in astrogliosis and glial cell‐derived neurotrophic factor/ brain‐derived neurotrophic factor expression in injured brain and astrocytes. These results indicate that DJ‐1 deficiency causes defects in astrocyte‐mediated repair of brain damage, which may contribute to the development of PD.