Antisense vimentin cDNA combined with chondroitinase ABC reduces glial scar and cystic cavity formation following spinal cord injury in rats

The formation of glial scar and cystic cavities restricts axon regeneration after spinal cord injury. Chondroitin sulphate proteoglycans (CSPGs) are regarded as the prominent inhibitory molecules in the glial scar, and their inhibitory effects may be abolished in part by chondroitinase ABC (ChABC), which can digest CSPGs. CSPGs are secreted mostly by reactive astrocytes, which form dense scar tissues. The intermediate filament protein vimentin underpins the cytoskeleton of reactive astrocytes. Previously we have shown that retroviruses carrying full-length antisense vimentin cDNA reduce reactive gliosis. Here we administered both antisense vimentin cDNA and ChABC to hemisected rat spinal cords. Using RT-PCR, Western blotting and immunohistochemistry, we found that the combined treatment reduced the formation of glial scar and cystic cavities through degrading CSPGs molecules and inhibiting intermediate filament proteins. The modified intra- and extra-cellular architecture may alter the physical and biochemical characteristics of the scar, and the combined therapy might be used to inhibit glial scar formation.     

RGMa inhibition promotes axonal growth and recovery after spinal cord injury

Repulsive guidance molecule (RGM) is a protein implicated in both axonal guidance and neural tube closure. We report RGMa as a potent inhibitor of axon regeneration in the adult central nervous system (CNS). RGMa inhibits mammalian CNS neurite outgrowth by a mechanism dependent on the activation of the RhoA-Rho kinase pathway. RGMa expression is observed in oligodendrocytes, myelinated fibers, and neurons of the adult rat spinal cord and is induced around the injury site after spinal cord injury. We developed an antibody to RGMa that efficiently blocks the effect of RGMa in vitro. Intrathecal administration of the antibody to rats with thoracic spinal cord hemisection results in significant axonal growth of the corticospinal tract and improves functional recovery. Thus, RGMa plays an important role in limiting axonal regeneration after CNS injury and the RGMa antibody offers a possible therapeutic agent in clinical conditions characterized by a failure of CNS regeneration.     

Transgenic inhibition of astroglial NF-κB leads to increased axonal sparing and sprouting following spinal cord injury

We previously showed that Nuclear Factor κB (NF-κB) inactivation in astrocytes leads to improved functional recovery following spinal cord injury (SCI). This correlated with reduced expression of pro-inflammatory mediators and chondroitin sulfate proteoglycans, and increased white matter preservation. Hence we hypothesized that inactivation of astrocytic NF-κB would create a more permissive environment for axonal sprouting and regeneration. We induced both contusive and complete transection SCI in GFAP-Inhibitor of κB-dominant negative (GFAP-IκBα-dn) and wild-type (WT) mice and performed retrograde [fluorogold (FG)] and anterograde [biotinylated dextran amine (BDA)] tracing 8 weeks after injury. Following contusive SCI, more FG-labeled cells were found in motor cortex, reticular formation, and raphe nuclei of transgenic mice. Spared and sprouting BDA-positive corticospinal axons were found caudal to the lesion in GFAP-IκBα-dn mice. Higher numbers of FG-labeled neurons were detected immediately rostral to the lesion in GFAP-IκBα-dn mice, accompanied by increased expression of synaptic and axonal growth-associated molecules. After transection, however, no FG-labeled neurons or BDA-filled axons were found rostral and caudal to the lesion, respectively, in either genotype. These data demonstrated that inhibiting astroglial NF-κB resulted in a growth-supporting terrain promoting sparing and sprouting, rather than regeneration, of supraspinal and propriospinal circuitries essential for locomotion, hence contributing to the improved functional recovery observed after SCI in GFAP-IκBα-dn mice.     

CSPGs在大鼠脊髓损伤后的表达及其与GFAP的关系

目的:观察脊髓损伤后CSPGs的表达及其与GFAP的关系。方法:成年雄性SD大鼠25只,随机分为对照组和损伤组,损伤组分脊髓挤压损伤后0h、72h、1w、4w组,运用免疫荧光双重染色方法观察CSPGs与GFAP的表达。结果:挤压伤后损伤部位的CSPGs和GFAP的表达均增高,但二者的变化趋势并不一样。其中CSPGs从损伤后表达开始增高,此后一直增加,并在1w至4w时逐渐稳定,主要分布逐渐集中于损伤部位;星形胶质细胞的免疫反应也逐渐增加,其分布逐渐集中于损伤区域的边缘,逐渐形成胶质瘢痕界膜。损伤1w至4w,损伤区域内几乎没有了星形胶质细胞表达,但仍留有大量的CSPGs。结论:早期抑制星形胶质细胞分泌CSPGs,可以防止在损伤部位沉积大量的CSPGs,从而减小其对再生纤维的抑制作用。     

Differential expression of several molecules of the extracellular matrix in functionally and developmentally distinct regions of rat spinal cord

We have examined the regional distribution of several chondroitin sulfate proteoglycans (neurocan, brevican, versican, aggrecan, phosphacan), of their glycosaminoglycan moieties, and of tenascin-R in the spinal cord of adult rat. The relationships of these molecules with glial and neuronal populations, identified with appropriate markers, were investigated by using multiple fluorescence labeling combined with confocal microscopy. The results showed that the distribution of the examined molecules was similar at all spinal cord levels but displayed area-specific differences along the dorso-ventral axis, delimiting functionally and developmentally distinct areas. In the gray matter, laminae I and II lacked perineuronal nets (PNNs) of extracellular matrix and contained low levels of chondroitin sulfate glycosaminoglycans (CS-GAGs), brevican, and tenascin-R, possibly favoring the maintenance of local neuroplastic properties. Conversely, CS-GAGs, brevican, and phosphacan were abundant, with numerous thick PNNs, in laminae III-VIII and X. Motor neurons (lamina IX) were surrounded by PNNs that contained all molecules investigated but displayed various amounts of CS-GAGs. Double-labeling experiments showed that the presence of PNNs could not be unequivocally related to specific classes of neurons, such as motor neurons or interneurons identified by their expression of calcium-binding proteins (parvalbumin, calbindin, calretinin). However, a good correlation was found between PNNs rich in CS-GAGs and the neuronal expression of the Kv3.1b subunit of the potassium channel, a marker of fast-firing neurons. This observation confirms the correlation between the electrophysiological properties of these neurons and the specific composition of their microenvironment.     

Adoptive transfer of Th1-conditioned lymphocytes promotes axonal remodeling and functional recovery after spinal cord injury

The role of T lymphocytes in central nervous system (CNS) injuries is controversial, with inconsistent results reported concerning the effects of T-lymphocyte transfer on spinal cord injury (SCI). Here, we demonstrate that a specific T-lymphocyte subset enhances functional recovery after contusion SCI in mice. Intraperitoneal adoptive transfer of type 1 helper T (Th1)-conditioned cells 4 days after SCI promoted recovery of locomotor activity and tactile sensation and concomitantly induced regrowth of corticospinal tract and serotonergic fibers. However, neither type 2 helper T (Th2)- nor IL-17-producing helper T (Th17)-conditioned cells had such effects. Activation of microglia and macrophages were observed in the spinal cords of Th1-transfered mice after SCI. Specifically, M2 subtype of microglia/macrophages was upregulated after Th1 cell transfer. Neutralization of interleukin 10 secreted by Th1-conditioned cells significantly attenuated the beneficial effects by Th1-conditioned lymphocytes after SCI. We also found that Th1-conditioned lymphocytes secreted significantly higher levels of neurotrophic factor, neurotrophin 3 (NT-3), than Th2- or Th17-conditioned cells. Thus, adoptive transfer of pro-inflammatory Th1-conditioned cells has neuroprotective effects after SCI, with prospective implications in immunomodulatory treatment of CNS injury.     

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.     

硫酸软骨素酶ABC治疗减少大鼠脊髓损伤引起的酪氨酸蛋白激酶A4的表达

本研究旨在探讨大鼠脊髓损伤(spinal cord impairment,SCI)后硫酸软骨素酶ABC (chondroitinase ABC,ChABC)对酪氨酸蛋白激酶A4 (ephrin A4,EphA4)表达变化的影响.选取成年雌性SD大鼠,随机分为假手术组、生理盐水(NS)组和ChABC组.NS组和ChABC...     

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.     

EphA4 blockers promote axonal regeneration and functional recovery following spinal cord injury in mice

Upregulation and activation of developmental axon guidance molecules, such as semaphorins and members of the Eph receptor tyrosine kinase family and their ligands, the ephrins, play a role in the inhibition of axonal regeneration following injury to the central nervous system. Previously we have demonstrated in a knockout model that axonal regeneration following spinal cord injury is promoted in the absence of the axon guidance protein EphA4. Antagonism of EphA4 was therefore proposed as a potential therapy to promote recovery from spinal cord injury. To further assess this potential, two soluble recombinant blockers of EphA4, unclustered ephrin-A5-Fc and EphA4-Fc, were examined for their ability to promote axonal regeneration and to improve functional outcome following spinal cord hemisection in wildtype mice. A 2-week administration of either of these blockers following spinal cord injury was sufficient to promote substantial axonal regeneration and functional recovery by 5 weeks following injury. Both inhibitors produced a moderate reduction in astrocytic gliosis, indicating that much of the effect of the blockers may be due to promotion of axon growth. These studies provide definitive evidence that soluble inhibitors of EphA4 function offer considerable therapeutic potential for the treatment of spinal cord injury and may have broader potential for the treatment of other central nervous system injuries.     

Glycan-dependent binding of galectin-1 to neuropilin-1 promotes axonal regeneration after spinal cord injury

Following spinal cord injury (SCI), semaphorin 3A (Sema3A) prevents axonal regeneration through binding to the neuropilin-1 (NRP-1)/PlexinA4 receptor complex. Here, we show that galectin-1 (Gal-1), an endogenous glycan-binding protein, selectively bound to the NRP-1/PlexinA4 receptor complex in injured neurons through a glycan-dependent mechanism, interrupts the Sema3A pathway and contributes to axonal regeneration and locomotor recovery after SCI. Although both Gal-1 and its monomeric variant contribute to de-activation of microglia, only high concentrations of wild-type Gal-1 (which co-exists in a monomer–dimer equilibrium) bind to the NRP-1/PlexinA4 receptor complex and promote axonal regeneration. Our results show that Gal-1, mainly in its dimeric form, promotes functional recovery of spinal lesions by interfering with inhibitory signals triggered by Sema3A binding to NRP-1/PlexinA4 complex, supporting the use of this lectin for the treatment of SCI patients.     

Overexpression of HuD, but not of its truncated form HuD I+II, promotes GAP-43 gene expression and neurite outgrowth in PC12 cells in the absence of nerve growth factor

We have previously shown that the RNA-binding protein HuD binds to a regulatory element in the growth-associated protein (GAP)-43 mRNA and that this interaction involves its first two RNA recognition motifs (RRMs). In this study, we investigated the functional significance of this interaction by overexpression of human HuD protein (pcHuD) or its truncated form lacking the third RRM (pcHuD I+II) in PC12 cells. Morphological analysis revealed that pcHuD cells extended short neurites containing GAP-43-positive growth cones in the absence of nerve growth factor (NGF). These processes also contained tubulin and F-actin filaments but were not stained with antibodies against neurofilament M protein. In correlation with this phenotype, pcHuD cells contained higher levels of GAP-43 without changes in levels of other NGF-induced proteins, such as SNAP-25 and tau. In mRNA decay studies, HuD stabilized the GAP-43 mRNA, whereas HuD I+II did not have any effect either on GAP-43 mRNA stability or on the levels of GAP-43 protein. Likewise, pcHuD I+II cells showed no spontaneous neurite outgrowth and deficient outgrowth in response to NGF. Our results indicate that HuD is sufficient to increase GAP-43 gene expression and neurite outgrowth in the absence of NGF and that the third RRM in the protein is critical for this function.     

Autophagy induced by Schwann cell-derived exosomes promotes recovery after spinal cord injury in rats

Biotechnology Letters - Spinal cord injury (SCI) is catastrophic to humans and society. However, there is currently no effective treatment for SCI. Autophagy is known to serve critical roles in...     

Overexpression of bone morphogenetic protein 7 reduces oligodendrocytes loss and promotes functional recovery after spinal cord injury

Spinal cord injury (SCI), as a severe disease with no effective therapeutic measures, has always been a hot topic for scientists. Bone morphogenetic protein 7 (BMP7), as a multifunctional cytokine, has been reported to exert protective effects on the nervous system. The present study aimed to investigate the neuroprotective effect and the potential mechanisms of BMP7 on rats that suffered SCI. Rat models of SCI were established by the modified Allen^'s method. Adeno-associated virus (AAV) was injected at T9 immediately before SCI to overexpress BMP7. Results showed that the expression of BMP7 decreased in the injured spinal cords that were at the same time demyelinated. AAV-BMP7 partly reversed oligodendrocyte (OL) loss, and it was beneficial to maintain the normal structure of myelin. The intervention group showed an increase in the number of axons and Basso-Beattie-Bresnahan scores. Moreover, double-labelled immunofluorescence images indicated p-Smad1/5/9 and p-STAT3 in OLs induced by BMP7 might be involved in the protective effects of BMP7. These findings suggest that BMP7 may be a feasible therapy for SCI to reduce demyelination and promote functional recovery.     

Increase in Neurite Formation and Acetylene line Release by Transfection of Growth-Associated Protein-43 cDNA into NG108–15 Cells

Abstract: We previously reported that growth-associated protein-43 (GAP-43) could be involved in the maintenance of elongated neurites and that a decline in protein kinase C activity may be involved in accumulation of GAP-43. In the present study, to clarify the functional significance of GAP-43 for neurite maintenance and acetylcholine (ACh) release, we prepared NG-G11 cells by transfection of GAP-43 cDNA into NG108-15 cells. NG-G11 cells expressed GAP-43 mRNA at levels approximately twice that in nontransfected or vector-transfected cells under control conditions and after treatment with dibutyryl cyclic AMP (diBu-cAMP) or 12-O-tetradecanoylphorbol 13-acetate (TPA) plus diBu-cAMP. Neurite outgrowth after addition of diBu-cAMP was greater in NG-G11 than in control cells. In NG-G11 cells, neurites elongated by treatment with diBu-cAMP for 72 h were maintained after removal of the drug. Treatment with TPA plus diBu-cAMP for 24 h induced neurite outgrowth in NG-G11 cells, although control cells required 72 h. Depolarization by 50 m M KCI induced ACh release in both NG-G11 and control cells treated with diBu-cAMP or TPA/diBu-cAMP. Although removal of the drugs following diBu-cAMP treatment reversed ACh release to nontreated levels in control cells, a high-K⁺-induced level of ACh release remained in NG-G11 cells after removal of diBu-cAMP. ACh release induced by TPA plus diBu-cAMP for 24 h was further enhanced after removal of the drugs in NG-G11 cells, but it was not seen in control cells. These results suggest that levels of GAP-43 mRNA are correlated with neurite maintenance and the level of ACh release. Thus, GAP-43 may be involved in neuronal differentiation in NG108-15 cells.     

Post-trauma Lipitor treatment prevents endothelial dysfunction, facilitates neuroprotection, and promotes locomotor recovery following spinal cord injury

We have previously reported neuroprotection in spinal cord injury (SCI) by Lipitor [atorvastatin (AT)]-pre-treatment. Though informative, pre-treatment studies find only limited clinical application as trauma occurrence is unpredictable. Therefore, this study investigates the efficacy of AT treatment post-SCI. In a rat model of contusion-SCI resulting in complete hindlimb paralysis, AT treatment (5 mg/kg; gavage) was begun 2, 4, or 6 h post-SCI followed by a once daily dose thereafter for 6 weeks. While the placebo vehicle (VHC)-SCI rats showed substantial functional deficit, AT-SCI animals exhibited significant functional recovery. AT diminished injury-induced blood-spinal cord barrier (BSCB) dysfunction with significantly reduced infiltration and tumor necrosis factor-alpha/interleukin-1beta/inducible nitric oxide synthase expression at site of injury. BSCB protection in AT-SCI was attributable to attenuated matrix metalloproteinase-9 (MMP9) expression - a central player in BSCB disruption. Furthermore, endothelial MMP9 expression was found to be RhoA/ROCK pathway-mediated and regulated by AT through an isoprenoid-dependent mechanism. Attenuation of these early inflammatory events reduced secondary damage. Significant reduction in axonal degeneration, myelin degradation, gliosis, and neuronal apoptosis with resultant enhancement in tissue sparing was observed in AT-SCI compared with VHC-SCI. In summary, this novel report presenting the efficacy of post-injury AT treatment might be of critical therapeutic value as effective treatments are currently unavailable for SCI.     

Melatonin attenuates methamphetamine-induced reduction of tyrosine hydroxylase, synaptophysin and growth-associated protein-43 levels in the neonatal rat brain

Methamphetamine (METH) is a most commonly abused drug which damages nerve terminals by causing formation of reactive oxygen species (ROS), apoptosis, and finally neuronal damage. Fetal exposure to neurotoxic METH causes significant behavioral effects. The developing fetus is substantially deficient in most antioxidative enzymes, and may therefore be at high risk from both endogenous and drug-enhanced oxidative stress. Little is known about the effects of METH on vesicular proteins such as synaptophysin and growth-associated protein 43 (GAP-43) in the immature brain. The present study attempted to investigate the effects of METH-induced neurotoxicity in the dopaminergic system of the neonatal rat brain. Neonatal rats were subcutaneously exposed to 5–10 mg/kg METH daily from postnatal day 4–10 for 7 consecutive days. The results showed that tyrosine hydroxylase enzyme levels were significantly decreased in the dorsal striatum, prefrontal cortex, nucleus accumbens and substantia nigra, synaptophysin levels decreased in the striatum and prefrontal cortex and growth-associated protein-43 (GAP-43) levels significantly decreased in the nucleus accumbens of neonatal rats. Pretreatment with 2 mg/kg melatonin 30 min prior to METH administration prevented METH-induced reduction in tyrosine hydroxylase, synaptophysin and growth-associated protein-43 protein levels in different brain regions. These results suggest that melatonin provides a protective effect against METH-induced nerve terminal degeneration in the immature rat brain probably via its antioxidant properties.     

MiR-92b-3p promotes neurite growth and functional recovery via the PTEN/AKT pathway in acute spinal cord injury

Emerging evidence indicates that microRNAs play an important role in neural remodeling, including neurite growth, after acute spinal cord injury (ASCI). This study aims to identify the mechanism by which miR-92b-3p regulates neurite growth in vivo and in vitro. Adult Sprague–Dawley rats were selected to establish the ASCI model, and the expressions of miR-92b-3p and phosphate and tensin homolog deleted on chromosome ten (PTEN) were quantified at different time points. The interaction between miR-92b-3p and PTEN was further detected in the PC12 cell line and dual-luciferase reporter assay. Neurite growth proteins (GAP43 and NF-200) were assessed by western blotting after miR-92b-3p mimics treatment. The PTEN/AKT pathway-related proteins and their roles in miR-92b-3p regulation were also identified using western blotting and immunofluorescence in vitro through LY294002, an AKT inhibitor. The effect of miR-92b-3p was further determined in vivo according to the Basso-Beattie-Bresnahan (BBB) Scale and GAP43 and NF-200 expressions. miR-92b-3p was downregulated after ASCI, while PTEN showed a simultaneous opposing trend. Overexpression of miR-92b-3p downregulated PTEN expression and promoted phosphorylation of AKT, as well as the expression of GAP43 and NF-200 in PC12 cells. Furthermore, the dual-luciferase reporter assay revealed that miR-92b-3p exerted its effect by targeting PTEN's 3ʹ-untranslated regions and that this effect could be counteracted by AKT phosphorylation blocker LY294002 through western blotting and immunofluorescence. Moreover, miR-92b-3p could also improve the BBB scale as well as GAP43 and NF-200 expression levels in vivo. Collectively, these results indicate that miR-92b-3p promotes neurite growth and functional recovery through the PTEN/AKT pathway in ASCI.