Triad1 regulates the expression and distribution of EHD1 contributing to the neurite outgrowth of neurons after spinal cord injury

Traumatic spinal cord injury is a common and severe complication after an accident. As we all know that neurite outgrowth of neurons is difficult after a spinal cord injury. Endosome system is associated with cargoes transportation and contributes in promoting the neuronal capability for neurite outgrowth. EH domain-containing protein 1 (EHD1) transports proteins through the endosome system, especially in the recycling endosomes and regulating the neurite outgrowth. In mammalian cells, the involvement of the ubiquitin-proteasome system in endosomal sorting has been well established. Two RING fingers and a DRIL (double RING finger-linked) 1 (Triad1) plays an important role in membrane trafficking and its mutant results in the wrong accumulation of receptors in endosomes and plasma membrane. In this current study, we reasonably integrated the results of the above research and investigated the regulating function of Triad1 to EHD1 following the spinal cord injury. We characterized the upregulated expression and distribution of Triad1 and EHD1 in the neurons after SCI and declared the interaction between Triad1 with EHD1 both in vitro and in vivo. Triad1 regulated the interaction between itself and the full-length or EH domain of EHD1, which influenced the neurite outgrowth of PC12 cells. Our data delineate a novel interaction between Triad1 and EHD1 that may contribute to the regulation of neurite outgrowth for neurons after the spinal cord injury.     

Stem Cells Downregulate the Elevated Levels of Tissue Plasminogen Activator in Rats After Spinal Cord Injury

We investigated the involvement of tPA after SCI in rats and effect of treatment with human umbilical cord blood derived stem cells. tPA expression and activity were determined in vivo after SCI in rats and in vitro in rat embryonic spinal neurons in response to injury with staurosporine, hydrogen peroxide and glutamate. The activity and/or expression of tPA increased after SCI and reached peak levels on day 21 post-SCI. Notably, the tPA mRNA activity was upregulated by 310-fold compared to controls on day 21 post-SCI. As expected, MBP expression is minimal at the time of peak tPA activity and vice versa. Implantation of hUCB after SCI resulted in the downregulation of elevated tPA activity/expression in vivo in rats as well as in vitro in spinal neurons. Our results demonstrated the involvement of tPA in the secondary pathogenesis after SCI as well as the therapeutic potential of hUCB.     

Effects of dibutyryl cyclic-AMP on survival and neuronal differentiation of neural stem/progenitor cells transplanted into spinal cord injured rats

Neural stem/progenitor cells (NSPCs) have great potential as a cell replacement therapy for spinal cord injury. However, poor control over transplant cell differentiation and survival remain major obstacles. In this study, we asked whether dibutyryl cyclic-AMP (dbcAMP), which was shown to induce up to 85% in vitro differentiation of NSPCs into neurons would enhance survival of transplanted NSPCs through prolonged exposure either in vitro or in vivo through the controlled release of dbcAMP encapsulated within poly(lactic-co-glycolic acid) (PLGA) microspheres and embedded within chitosan guidance channels. NSPCs, seeded in fibrin scaffolds within the channels, differentiated in vitro to betaIII-tubulin positive neurons by immunostaining and mRNA expression, in response to dbcAMP released from PLGA microspheres. After transplantation in spinal cord injured rats, the survival and differentiation of NSPCs was evaluated. Untreated NSPCs, NSPCs transplanted with dbcAMP-releasing microspheres, and NSPCs pre-differentiated with dbcAMP for 4 days in vitro were transplanted after rat spinal cord transection and assessed 2 and 6 weeks later. Interestingly, NSPC survival was highest in the dbcAMP pre-treated group, having approximately 80% survival at both time points, which is remarkable given that stem cell transplantation often results in less than 1% survival at similar times. Importantly, dbcAMP pre-treatment also resulted in the greatest number of in vivo NSPCs differentiated into neurons (37±4%), followed by dbcAMP-microsphere treated NSPCs (27±14%) and untreated NSPCs (15±7%). The reverse trend was observed for NSPC-derived oligodendrocytes and astrocytes, with these populations being highest in untreated NSPCs. This combination strategy of stem cell-loaded chitosan channels implanted in a fully transected spinal cord resulted in extensive axonal regeneration into the injury site, with improved functional recovery after 6 weeks in animals implanted with pre-differentiated stem cells in chitosan channels.     

Spatiotemporal Expression of Dexras1 After Spinal Cord Transection in Rats

Dexras1, a brain-enriched member of the Ras subfamily of GTPases, as a novel physiologic nitric oxide (NO) effector, anchor neuronal nitric oxide synthase (nNOS) that increased after spinal cord injury (SCI), to specific targets to enhance NO signaling, and is strongly and rapidly induced during treatment with dexamethasone. It is unknown how the central nervous system (CNS) trauma affects the expression of Dexras1. Here we used spinal cord transection (SCT) model to detect expression of Dexras1 at mRNA and protein level in spinal cord homogenates by real-time PCR and Western blot analysis. The results showed that Dexras1 mRNA upregulated at 3 day, 5 day, and 7 day significantly (P < 0.05) that was consistent with the protein level except at 7 day. Immunofluorescence revealed that both neurons and glial cells showed Dexras1 immunoreactivivty (IR) around SCT site, but the proportion is different. Importantly, injury-induced expression of Dexras1 was co-labeled by caspase-3 (apoptotic marker) and Tau-1 (marker for pathological oligodendrocyte). Furthermore, colocalization of Dexras1, carboxy-terminal PSD95/DLG/ZO-1 (PDZ) ligand of nNOS (CAPON) and nNOS was observed in neurons and glial cells, supporting the existence of ternary complexes in this model. Thus, the results that the transient high expression of Dexras1 which localized in apoptotic neurons and pathological oligodendrocytes might provide new insight into the secondary response after SCT. Xin Li, Chun Cheng, and Min Fei contributed equally to this work.     

Permissive role of Cdc2 activity induced from astrocytes in neurite outgrowth

Following spinal cord injury, glial cells are recognized as major environmental factors hampering axon's regenerative responses. However, recent studies suggested that, in certain circumstances, reactive astrocytes may have a permissive role for axonal regeneration and functional recovery. Here, we report that Cdc2 activation in astrocytes is positively linked to axon growth. Cdc2 was strongly, but transiently, induced from reactive astrocytes within and around the injury cavity. Cdc2 levels in primary, non‐neuronal cells prepared from injured spinal cord were up‐regulated by extending the pre‐injury period. Cdc2‐mediated vimentin phosphorylation was strongly induced in astrocytes after long‐term culture (7 days, LTC) as compared with short‐term culture (3 days, STC). Induction levels of phospho‐vimentin in LTC astrocytes were positively associated with increased neurite outgrowth in co‐cultured dorsal root ganglion neurons. β3 integrin mRNA was induced in LTC astrocytes and activation of β3 integrin was regulated by Cdc2 activity. Furthermore, genetic depletion and pharmacological blockade experiments demonstrate that activation of Cdc2 and β3 integrin in LTC astrocytes is required for neurite outgrowth. Our data suggest that the Cdc2 pathway may play an important role in determining phenotypic expression of astrocytes such that astrocytes provide permissive environments for axonal regeneration following spinal cord injury.     

Expression and Cellular Distribution of Ubiquitin in Response to Injury in the Developing Spinal Cord of Monodelphis domestica

Ubiquitin, an 8.5 kDa protein associated with the proteasome degradation pathway has been recently identified as differentially expressed in segment of cord caudal to site of injury in developing spinal cord. Here we describe ubiquitin expression and cellular distribution in spinal cord up to postnatal day P35 in control opossums (Monodelphis domestica) and in response to complete spinal transection (T10) at P7, when axonal growth through site of injury occurs, and P28 when this is no longer possible. Cords were collected 1 or 7 days after injury, with age-matched controls and segments rostral to lesion were studied. Following spinal injury ubiquitin levels (western blotting) appeared reduced compared to controls especially one day after injury at P28. In contrast, after injury mRNA expression (qRT-PCR) was slightly increased at P7 but decreased at P28. Changes in isoelectric point of separated ubiquitin indicated possible post-translational modifications. Cellular distribution demonstrated a developmental shift between earliest (P8) and latest (P35) ages examined, from a predominantly cytoplasmic immunoreactivity to a nuclear expression; staining level and shift to nuclear staining was more pronounced following injury, except 7 days after transection at P28. After injury at P7 immunostaining increased in neurons and additionally in oligodendrocytes at P28. Mass spectrometry showed two ubiquitin bands; the heavier was identified as a fusion product, likely to be an ubiquitin precursor. Apparent changes in ubiquitin expression and cellular distribution in development and response to spinal injury suggest an intricate regulatory system that modulates these responses which, when better understood, may lead to potential therapeutic targets.     

FGF-2 Up-regulation and proliferation of neural progenitors in the regenerating amphibian spinal cord in vivo

Regeneration of the spinal cord occurs spontaneously in adult urodele amphibians. The key cells in this regenerative process appear to be the ependymal cells that following injury migrate and proliferate to form the ependymal tube from which the spinal cord regenerates. Very little is known about the signal(s) that initiates and maintains the proliferative response of these cells. Fibroblast growth factor 2 (FGF-2) has been shown to play a role in maintaining neural progenitor cell cycling in vitro and may be important for neuronal survival and axonal growth after injury. We have investigated its role in regeneration of the spinal cord in vivo following tail amputation in the adult salamander, Pleurodeles waltl. We show that only the low-molecular-weight form of FGF-2 is found in Pleurodeles and that in the normal cord it is expressed in a subset of neurons, but is hardly detectable in ependymal cells. Tail amputation results in induction of FGF-2 in the ependymal cells of the regenerating structure, and later in regeneration FGF-2 is up-regulated in some newborn neurons. FGF-2 pattern of expression in the ependymal tube parallels that of proliferation. Furthermore, exogenous FGF-2 significantly increases ependymal cell proliferation in vivo. Overall our results strongly support the view that one important role of FGF-2 during spinal cord regeneration in Pleurodeles is to induce proliferation of neural progenitor cells.     

Expression of neural cell adhesion molecule in spinal cords following a complete transection

Neural cell adhesion molecule (NCAM) regulates tissue organization during development and in the adult. NCAM upregulation occurs after an injury to brains and sciatic nerves. However, little is known about NCAM expression after spinal cord injury (SCI). By using a complete spinal cord transection with a 5 mm tissue removal, an increase in the NCAM level is detected in spinal cord stumps proximal and distal to the transection site at 1 d and 3 d post injury, while its expression at 8 d is declined to a lower level than that observed in sham-operated spinal cords. The strong NCAM expression is present in motor neurons at 3 d post transection whereas the intensive NCAM immunostaining is localized in dorsal sensory and corticospinal fiber tracts at 8 d following injury. Collectively, NCAM level is elevated and strongly expressed in dorsal fiber tracts after SCI, implying that the endogenous process for spinal cord regeneration may take place after SCI.     

Dual regulation of microglia and neurons by Astragaloside IV‐mediated mTORC1 suppression promotes functional recovery after acute spinal cord injury

Inflammation and neuronal apoptosis contribute to the progression of secondary injury after spinal cord injury (SCI) and are targets for SCI therapy; autophagy is reported to suppress apoptosis in neuronal cells and M2 polarization may attenuate inflammatory response in microglia, while both are negatively regulated by mTORC1 signalling. We hypothesize that mTORC1 suppression may have dual effects on inflammation and neuronal apoptosis and may be a feasible approach for SCI therapy. In this study, we evaluate a novel inhibitor of mTORC1 signalling, Astragaloside IV (AS‐IV), in vitro and in vivo. Our results showed that AS‐IV may suppress mTORC1 signalling both in neuronal cells and microglial cells in vitro and in vivo. AS‐IV treatment may stimulate autophagy in neuronal cells and protect them against apoptosis through autophagy regulation; it may also promote M2 polarization in microglial cells and attenuate neuroinflammation. In vivo, rats were intraperitoneally injected with AS‐IV (10 mg/kg/d) after SCI, behavioural and histological evaluations showed that AS‐IV may promote functional recovery in rats after SCI. We propose that mTORC1 suppression may attenuate both microglial inflammatory response and neuronal apoptosis and promote functional recovery after SCI, while AS‐IV may become a novel therapeutic medicine for SCI.     

Phenotype of distinct primary sensory afferent subpopulations and caspase-3 expression following axotomy

Specific sensory neuronal subpopulations show contrasting responses to peripheral nerve injury, as shown by the axotomy-induced death of many cutaneous sensory neurons whilst muscular sensory afferents survive an identical insult. We used a novel combination of retrograde neuronal tracing with immunohistochemistry and laser microdissection techniques, in order to describe the neurochemistry of medial gastrocnemius (muscular sensory afferents) and sural (cutaneous sensory afferents) branches of the rat sciatic nerve and relate this to the pro-apoptotic caspase-3 gene expression following nerve transection. Our results demonstrated distinctions in medial gastrocnemius and sural neuron populations with the most striking difference in the respective proportions of isolectin B4 (IB4) staining neurons (3.7 V 32.8%). The mean neuronal area of the medial gastrocnemius (MG) neurons was larger than that of the sural (SUR) neurons (1,070.8 V 646.2 μm2) and each phenotypic group was significantly smaller in sural neurons than in MG neurons. At 1 week post-axotomy, MG neurons markedly downregulated caspase-3, whilst SUR neurons upregulated caspase-3 gene expression; this may be attributable to the differing IB4-positive composition of the subpopulations. These findings provide further clarification in the understanding of two distinct neuronal populations used increasingly in nerve injury models.     

p53- and Bax-Mediated Apoptosis in Injured Rat Spinal Cord

Spinal cord injury (SCI) induces a series of endogenous biochemical changes that lead to secondary degeneration, including apoptosis. p53-mediated mitochondrial apoptosis is likely to be an important mechanism of cell death in spinal cord injury. However, the signaling cascades that are activated before DNA fragmentation have not yet been determined. DNA damage-induced, p53-activated neuronal cell death has already been identified in several neurodegenerative diseases. To determine DNA damage-induced, p53-mediated apoptosis in spinal cord injury, we performed RT-PCR microarray and analyzed 84 DNA damaging and apoptotic genes. Genes involved in DNA damage and apoptosis were upregulated whereas anti-apoptotic genes were downregulated in injured spinal cords. Western blot analysis showed the upregulation of DNA damage-inducing protein such as ATM, cell cycle checkpoint kinases, 8-hydroxy-2′-deoxyguanosine (8-OHdG), BRCA2 and H2AX in injured spinal cord tissues. Detection of phospho-H2AX in the nucleus and release of 8-OHdG in cytosol were demonstrated by immunohistochemistry. Expression of p53 was observed in the neurons, oligodendrocytes and astrocytes after spinal cord injury. Upregulation of phospho-p53, Bax and downregulation of Bcl2 were detected after spinal cord injury. Sub-cellular distribution of Bax and cytochrome c indicated mitochondrial-mediated apoptosis taking place after spinal cord injury. In addition, we carried out immunohistochemical analysis to confirm Bax translocation into the mitochondria and activated p53 at Ser³⁹². Expression of APAF1, caspase 9 and caspase 3 activities confirmed the intrinsic apoptotic pathway after SCI. Activated p53 and Bax mitochondrial translocation were detected in injured spinal neurons. Taken together, the in vitro data strengthened the in vivo observations of DNA damage-induced p53-mediated mitochondrial apoptosis in the injured spinal cord.     

The mechanical properties of neonatal rat spinal cord in vitro,and comparisons with adult

A number of studies have investigated the mechanical properties of adult spinal cord under tension, however it is not known whether age has an effect on these properties. This is of interest to those aiming to understand the clinical differences between adults and children with spinal cord injury (e.g. severity and recovery), and those developing experimental or computational models for paediatric spinal cord injury. Entire spinal cords were freshly harvested from neonatal rats (14 days) and tested in vitro under uniaxial tension at a range of strain rates (0.2, 0.02, 0.002/s) to a range of strains (2%, 3.5%, 5%), with relaxation responses being recorded for 15 min. These mechanical properties were compared to previously reported data from similar experiments on adult rat spinal cords, and the peak stress and the stress after 15 min of relaxation were found to be significantly higher for spinal cords from adults than neonates (p<0.001). A non-linear viscoelastic model was developed and was observed to adequately predict the mechanical behaviour of this tissue. The model developed in this study may be of use in computational models of paediatric spinal cord. The significant differences between adult and neonatal spinal cord properties may explain the higher initial severity of spinal cord injury in children and may have implications for the development of experimental animal models for paediatric spinal cord injury, specifically for those aiming to match the injury severity with adult experimental models.     

Up-regulation of TRAF2 Suppresses Neuronal Apoptosis after Rat Spinal Cord Injury

Tumor necrosis factor receptor-associated factor 2 (TRAF2) for signal transduction of the cell death receptor is well established. However, the role of TRAF2 in spinal cord injury (SCI) remains unclear. In this study, we detected the dynamic change patterns of TRAF2 expression using an acute spinal cord contusion (SCC) model in adult rats. Western blot analysis and immunohistochemistry identified significant upregulation of TRAF2 after SCI. Double-immunofluorescent staining demonstrated that the upregulated TRAF2 was found predominantly in neurons. Moreover, colocalization of TRAF2 with active caspase-3/-8 was detected in NeuN-positive cells. In vitro, we analyzed the association of TRAF2 with active caspase-3/8 on PC12 cells by western blot analysis, which paralleled the in vivo data. Knockdown ofTRAF2 with siRNA demonstrated its probable anti-apoptotic role in the process of neuronal apoptosis after SCI. To summarize, we have revealed for the first time the temporal and spatial expression profile of TRAF2 in SCI. Our data suggest that upregulation of TRAF2 triggered by trauma plays an important role in suppressing neuronal apoptosis after SCI.     

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.     

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16 °C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 °C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.     

An analysis of dorsal root ganglia differentiation using three tissue culture systems

Summary The histogenesis of the dorsal root ganglia of chick embryos (ages 3 to 9 days) was followed in three different tissue culture systems. Organotypic explants included dorsal root ganglia connected to the lumbosacral segment of the spinal cord or isolated explants of the contralateral ganglia. Additionally, dissociated monolayer cultures of ganglia tissue were established. The gradual differentiation of progenitor neuroblasts into distinct populations of large ventrolateral and small dorsomedial neurons was observed in vivo and in vitro. Neurites developed after 3 days in the presence or absence of nerve growth factor in the medium. In contrast, autoradiographic analysis indicates that [³H]thymidine incorporation in neuronal cultures differed significantly from intact embryos. In vivo, the number of neuronal progenitor cells labeled with [³H]thymidine decreased in older embryos; in vitro, uptake of [³H]thymidine label was not observed in ganglionic progenitor cells regardless of the age of the donor embryo or the type of culture system. Lack of proliferation in ganglionic progenitor cells was not due to degeneration because vital staining and uptake of [³H]deoxyglucose indicated that neurons were metabolically active. Furthermore, the block in mitotic activity in vitro was limited to presumptive ganglionic neuronal cells. In the ependyma of the spinal cord segment connected to the dorsal root ganglia, neuronal progenitor cells were heavily labeled as were non-neuronal cells within both spinal cord and ganglia. Our results suggest that in vitro conditions can promote the differentiation of sensory neurons from early embryos (E3.5–4.5) without proliferation of progenitor cells.     

Rea regulates microglial polarization and attenuates neuronal apoptosis via inhibition of the NF-κB and MAPK signalings for spinal cord injury repair

Inflammation and neuronal apoptosis aggravate the secondary damage after spinal cord injury (SCI). Rehmannioside A (Rea) is a bioactive herbal extract isolated from Rehmanniae radix with low toxicity and neuroprotection effects. Rea treatment inhibited the release of pro-inflammatory mediators from microglial cells, and promoted M2 polarization in vitro, which in turn protected the co-cultured neurons from apoptosis via suppression of the NF-κB and MAPK signalling pathways. Furthermore, daily intraperitoneal injections of 80 mg/kg Rea into a rat model of SCI significantly improved the behavioural and histological indices, promoted M2 microglial polarization, alleviated neuronal apoptosis, and increased motor function recovery. Therefore, Rea is a promising therapeutic option for SCI and should be clinically explored.