Chx10+V2a interneurons in spinal motor regulation and spinal cord injury.

Chx10-expressing V2a (Chx10+V2a) spinal interneurons play a large role in the excitatory drive of motoneurons. Chemogenetic ablation studies have demonstrated the essential nature of Chx10+V2a interneurons in the regulation of locomotor initiation, maintenance, alternation, speed, and rhythmicity. The role of Chx10+V2a interneurons in locomotion and autonomic nervous system regulation is thought to be robust, but their precise role in spinal motor regulation and spinal cord injury have not been fully explored. The present paper reviews the origin, characteristics, and functional roles of Chx10+V2a interneurons with an emphasis on their involvement in the pathogenesis of spinal cord injury. The diverse functional properties of these cells have only been substantiated by and are due in large part to their integration in a variety of diverse spinal circuits. Chx10+V2a interneurons play an integral role in conferring locomotion, which integrates various corticospinal, mechanosensory, and interneuron pathways. Moreover, accumulating evidence suggests that Chx10+V2a interneurons also play an important role in rhythmic patterning maintenance, left-right alternation of central pattern generation, and locomotor pattern generation in higher order mammals, likely conferring complex locomotion. Consequently, the latest research has focused on postinjury transplantation and noninvasive stimulation of Chx10+V2a interneurons as a therapeutic strategy, particularly in spinal cord injury. Finally, we review the latest preclinical study advances in laboratory derivation and stimulation/transplantation of these cells as a strategy for the treatment of spinal cord injury. The evidence supports that the Chx10+V2a interneurons act as a new therapeutic target for spinal cord injury. Future optimization strategies should focus on the viability, maturity, and functional integration of Chx10+V2a interneurons transplanted in spinal cord injury foci.

Restoring mitochondrial cardiolipin homeostasis reduces cell death and promotes recovery after spinal cord injury.

Alterations in phospholipids have long been associated with spinal cord injury (SCI). However, their specific roles and signaling cascades in mediating cell death and tissue repair remain unclear. Here we investigated whether alterations of cardiolipin (CL), a family of mitochondrion-specific phospholipids, play a crucial role in mitochondrial dysfunction and neuronal death following SCI. Lipidomic analysis was used to determine the profile of CL alteration in the adult rat spinal cord following a moderate contusive SCI at the 10th thoracic (T10) level. Cellular, molecular, and genetic assessments were performed to determine whether CL alterations mediate mitochondrial dysfunction and neuronal death after SCI, and, if so, whether reversing CL alteration leads to neuroprotection after SCI. Using lipidomic analysis, we uncovered CL alterations at an early stage of SCI. Over 50 distinct CL species were identified, of which 50% showed significantly decreased abundance after SCI. The decreased CL species contained mainly polyunsaturated fatty acids that are highly susceptible to peroxidation. In parallel, 4-HNE, a lipid peroxidation marker, significantly increased after SCI. We found that mitochondrial oxidative stress not only induced CL oxidation, but also resulted in CL loss by activating cPLA2 to hydrolyze CL. CL alterations induced mitochondrial dysfunction and neuronal death. Remarkably, pharmacologic inhibition of CL alterations with XJB-5-131, a novel mitochondria-targeted electron and reactive oxygen species scavenger, reduced cell death, tissue damage and ameliorated motor deficits after SCI in adult rats. These findings suggest that CL alteration could be a novel mechanism that mediates injury-induced neuronal death, and a potential therapeutic target for ameliorating secondary SCI.

Interaction between Schwann cells and other cells during repair of peripheral nerve injury.

Peripheral nerve injury (PNI) is common and, unlike damage to the central nervous system injured nerves can effectively regenerate depending on the location and severity of injury. Peripheral myelinating glia, Schwann cells (SCs), interact with various cells in and around the injury site and are important for debris elimination, repair, and nerve regeneration. Following PNI, Wallerian degeneration of the distal stump is rapidly initiated by degeneration of damaged axons followed by morphologic changes in SCs and the recruitment of circulating macrophages. Interaction with fibroblasts from the injured nerve microenvironment also plays a role in nerve repair. The replication and migration of injury-induced dedifferentiated SCs are also important in repairing the nerve. In particular, SC migration stimulates axonal regeneration and subsequent myelination of regenerated nerve fibers. This mobility increases SC interactions with other cells in the nerve and the exogenous environment, which influence SC behavior post-injury. Following PNI, SCs directly and indirectly interact with other SCs, fibroblasts, and macrophages. In addition, the inter- and intracellular mechanisms that underlie morphological and functional changes in SCs following PNI still require further research to explain known phenomena and less understood cell-specific roles in the repair of the injured peripheral nerve. This review provides a basic assessment of SC function post-PNI, as well as a more comprehensive evaluation of the literature concerning the SC interactions with macrophages and fibroblasts that can influence SC behavior and, ultimately, repair of the injured nerve.

Laminin-coated multifilament entubulation, combined with Schwann cells and glial cell line-derived neurotrophic factor, promotes unidirectional axonal regeneration in a rat model of thoracic spinal cord hemisection.

Biomaterial bridging provides physical substrates to guide axonal growth across the lesion. To achieve efficient directional guidance, combinatory strategies using permissive matrix, cells and trophic factors are necessary. In the present study, we evaluated permissive effect of poly (acrylonitrile-co-vinyl chloride) guidance channels filled by different densities of laminin-precoated unidirectional polypropylene filaments combined with Schwann cells, and glial cell line-derived neurotrophic factor for axonal regeneration through a T10 hemisected spinal cord gap in adult rats. We found that channels with filaments significantly reduced the lesion cavity, astrocytic gliosis, and inflammatory responses at the graft-host boundaries. The laminin coated low density filament provided the most favorable directional guidance for axonal regeneration which was enhanced by co-grafting of Schwann cells and glial cell line-derived neurotrophic factor. These results demonstrate that the combinatorial strategy of filament-filled guiding scaffold, adhesive molecular laminin, Schwann cells, and glial cell line-derived neurotrophic factor, provides optimal topographical cues in stimulating directional axonal regeneration following spinal cord injury. This study was approved by Indiana University Institutional Animal Care and Use Committees (IACUC #:11011) on October 29, 2015.

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats.

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human Brown-Séquard syndrome is a common type of incomplete SCI caused by a lesion to one half of the spinal cord which results in paralysis and loss of proprioception on the same (or ipsilesional) side as the injury, and loss of pain and temperature sensation on the opposite (or contralesional) side. Adequate methodologies for producing a spinal cord lateral hemisection (HX) and assessing neurological impairments are essential to establish a reliable animal model of Brown-Séquard syndrome. Although lateral hemisection model plays a pivotal role in basic and translational research, standardized protocols for creating such a hemisection and assessing unilateralized function are lacking. The goal of this study is to describe step-by-step procedures to produce a rat spinal lateral HX at the 9th thoracic (T9) vertebral level. We, then, describe a combined behavior scale for HX (CBS-HX) that provides a simple and sensitive assessment of asymmetric neurological performance for unilateral SCI. The CBS-HX, ranging from 0 to 18, is composed of 4 individual assessments which include unilateral hindlimb stepping (UHS), coupling, contact placing, and grid walking. For CBS-HX, the ipsilateral and contralateral hindlimbs are assessed separately. We found that, after a T9 HX, the ipsilateral hindlimb showed impaired behavior function whereas the contralateral hindlimb showed substantial recovery. The CBS-HX effectively discriminated behavioral functions between ipsilateral and contralateral hindlimbs and detected temporal progression of recovery of the ipsilateral hindlimb. The CBS-HX components can be analyzed separately or in combination with other measures when needed. Although we only provided visual descriptions of the surgical procedures and behavioral assessments of a thoracic HX, the principle may be applied to other incomplete SCIs and at other levels of the injury.

Remodeling of lumbar motor circuitry remote to a thoracic spinal cord injury promotes locomotor recovery.

Retrogradely-transported neurotrophin signaling plays an important role in regulating neural circuit specificity. Here we investigated whether targeted delivery of neurotrophin-3 (NT-3) to lumbar motoneurons (MNs) caudal to a thoracic (T10) contusive spinal cord injury (SCI) could modulate dendritic patterning and synapse formation of the lumbar MNs. In vitro, Adeno-associated virus serotype two overexpressing NT-3 (AAV-NT-3) induced NT-3 expression and neurite outgrowth in cultured spinal cord neurons. In vivo, targeted delivery of AAV-NT-3 into transiently demyelinated adult mouse sciatic nerves led to the retrograde transportation of NT-3 to the lumbar MNs, significantly attenuating SCI-induced lumbar MN dendritic atrophy. NT-3 enhanced sprouting and synaptic formation of descending serotonergic, dopaminergic, and propriospinal axons on lumbar MNs, parallel to improved behavioral recovery. Thus, retrogradely transported NT-3 stimulated remodeling of lumbar neural circuitry and synaptic connectivity remote to a thoracic SCI, supporting a role for retrograde transport of NT-3 as a potential therapeutic strategy for SCI.

Inhibition of KLF7-Targeting MicroRNA 146b Promotes Sciatic Nerve Regeneration.

A previous study has indicated that Krüppel-like factor 7 (KLF7), a transcription factor that stimulates Schwann cell (SC) proliferation and axonal regeneration after peripheral nerve injury, is a promising therapeutic transcription factor in nerve injury. We aimed to identify whether inhibition of microRNA-146b (miR-146b) affected SC proliferation, migration, and myelinated axon regeneration following sciatic nerve injury by regulating its direct target KLF7. SCs were transfected with miRNA lentivirus, miRNA inhibitor lentivirus, or KLF7 siRNA lentivirus in vitro. The expression of miR146b and KLF7, as well as SC proliferation and migration, were subsequently evaluated. In vivo, an acellular nerve allograft (ANA) followed by injection of GFP control vector or a lentiviral vector encoding an miR-146b inhibitor was used to assess the repair potential in a model of sciatic nerve gap. miR-146b directly targeted KLF7 by binding to the 3'-UTR, suppressing KLF7. Up-regulation of miR-146b and KLF7 knockdown significantly reduced the proliferation and migration of SCs, whereas silencing miR-146b resulted in increased proliferation and migration. KLF7 protein was localized in SCs in which miR-146b was expressed in vivo. Similarly, 4 weeks after the ANA, anti-miR-146b increased KLF7 and its target gene nerve growth factor cascade, promoting axonal outgrowth. Closer analysis revealed improved nerve conduction and sciatic function index score, and enhanced expression of neurofilaments, P0 (anti-peripheral myelin), and myelinated axon regeneration. Our findings provide new insight into the regulation of KLF7 by miR-146b during peripheral nerve regeneration and suggest a potential therapeutic strategy for peripheral nerve injury.

AAV-KLF7 Promotes Descending Propriospinal Neuron Axonal Plasticity after Spinal Cord Injury.

DPSN axons mediate and maintain a variety of normal spinal functions. Unsurprisingly, DPSN tracts have been shown to mediate functional recovery following SCI. KLF7 could contribute to CST axon plasticity after spinal cord injury. In the present study, we assessed whether KLF7 could effectively promote DPSN axon regeneration and synapse formation following SCI. An AAV-KLF7 construct was used to overexpress KLF7. In vitro, KLF7 and target proteins were successfully elevated and axonal outgrowth was enhanced. In vivo, young adult C57BL/6 mice received a T10 contusion followed by an AAV-KLF7 injection at the T7-9 levels above the lesion. Five weeks later, overexpression of KLF7 was expressed in DPSN. KLF7 and KLF7 target genes (NGF, TrkA, GAP43, and P0) were detectably increased in the injured spinal cord. Myelin sparring at the lesion site, DPSN axonal regeneration and synapse formation, muscle weight, motor endplate morphology, and functional parameters were all additionally improved by KLF7 treatment. Our findings suggest that KLF7 promotes DPSN axonal plasticity and the formation of synapses with motor neurons at the caudal spinal cord, leading to improved functional recovery and further supporting the potential of AAV-KLF7 as a therapeutic agent for spinal cord injury.

KLF7-transfected Schwann cell graft transplantation promotes sciatic nerve regeneration.

Our former study demonstrated that Krüppel-like Factor 7 (KLF7) is a transcription factor that stimulates axonal regeneration after peripheral nerve injury. Currently, we used a gene therapy approach to overexpress KLF7 in Schwann cells (SCs) and assessed whether KLF7-transfected SCs graft could promote sciatic nerve regeneration. SCs were transfected by adeno-associated virus 2 (AAV2)-KLF7 in vitro. Mice were allografted by an acellular nerve (ANA) with either an injection of DMEM (ANA group), SCs (ANA+SCs group) or AAV2-KLF7-transfected SCs (ANA+KLF7-SCs group) to assess repair of a sciatic nerve gap. The results indicate that KLF7 overexpression promoted the proliferation of both transfected SCs and native SCs. The neurite length of the dorsal root ganglia (DRG) explants was enhanced. Several beneficial effects were detected in the ANA+KLF7-SCs group including an increase in the compound action potential amplitude, sciatic function index score, enhanced expression of PKH26-labeling transplant SCs, peripheral myelin protein 0, neurofilaments, S-100, and myelinated regeneration nerve. Additionally, HRP-labeled motoneurons in the spinal cord, CTB-labeled sensory neurons in the DRG, motor endplate density and the weight ratios of target muscles were increased by the treatment while thermal hyperalgesia was diminished. Finally, expression of KLF7, NGF, GAP43, TrkA and TrkB were enhanced in the grafted SCs, which may indicate that several signal pathways may be involved in conferring the beneficial effects from KLF7 overexpression. We concluded that KLF7-overexpressing SCs promoted axonal regeneration of the peripheral nerve and enhanced myelination, which collectively proved KLF-SCs as a novel therapeutic strategy for injured nerves.

Transcriptional and Epigenetic Regulation in Injury-Mediated Neuronal Dendritic Plasticity.

Injury to the nervous system induces localized damage in neural structures and neuronal death through the primary insult, as well as delayed atrophy and impaired plasticity of the delicate dendritic fields necessary for interneuronal communication. Excitotoxicity and other secondary biochemical events contribute to morphological changes in neurons following injury. Evidence suggests that various transcription factors are involved in the dendritic response to injury and potential therapies. Transcription factors play critical roles in the intracellular regulation of neuronal morphological plasticity and dendritic growth and patterning. Mounting evidence supports a crucial role for epigenetic modifications via histone deacetylases, histone acetyltransferases, and DNA methyltransferases that modify gene expression in neuronal injury and repair processes. Gene regulation through epigenetic modification is of great interest in neurotrauma research, and an early picture is beginning to emerge concerning how injury triggers intracellular events that modulate such responses. This review provides an overview of injury-mediated influences on transcriptional regulation through epigenetic modification, the intracellular processes involved in the morphological consequences of such changes, and potential approaches to the therapeutic manipulation of neuronal epigenetics for regulating gene expression to facilitate growth and signaling through dendritic arborization following injury.

Unilateral microinjection of acrolein into thoracic spinal cord produces acute and chronic injury and functional deficits.

Although lipid peroxidation has long been associated with spinal cord injury (SCI), the specific role of lipid peroxidation-derived byproducts such as acrolein in mediating damage remains to be fully understood. Acrolein, an α-ß unsaturated aldehyde, is highly reactive with proteins, DNA, and phospholipids and is considered as a second toxic messenger that disseminates and augments initial free radical events. Previously, we showed that acrolein increased following traumatic SCI and injection of acrolein induced tissue damage. Here, we demonstrate that microinjection of acrolein into the thoracic spinal cord of adult rats resulted in dose-dependent tissue damage and functional deficits. At 24h (acute) after the microinjection, tissue damage, motoneuron loss, and spinal cord swelling were observed on sections stained with Cresyl Violet. Luxol fast blue staining further showed that acrolein injection resulted in dose-dependent demyelination. At 8weeks (chronic) after the microinjection, cord shrinkage, astrocyte activation, and macrophage infiltration were observed along with tissue damage, neuron loss, and demyelination. These pathological changes resulted in behavioral impairments as measured by both the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale and grid walking analysis. Electron microscopy further demonstrated that acrolein induced axonal degeneration, demyelination, and macrophage infiltration. These results, combined with our previous reports, strongly suggest that acrolein may play a critical causal role in the pathogenesis of SCI and that targeting acrolein could be an attractive strategy for repair after SCI.

Sciatic nerve regeneration in KLF7-transfected acellular nerve allografts.

OBJECTIVE: Krüppel-like Factor 7 (KLF7) is a transcription factor that promotes axon regeneration in the central nervous system. Here, we assessed whether KLF7 stimulates regeneration after peripheral nerve injury. METHODS: C57BL/6 mice received an acellular nerve allograft (ANA) injected with either adeno-associated virus 2 (AAV2) vector or AAV2-KLF7 for sciatic nerve gap repair. After 4 weeks, KLF7 was detected by RT-PCR, western blot and immunohistochemistry in regenerated nerves. Axonal regeneration and functional recovery were examined by immunohistochemistry, Fluorogold (FG) and cholera toxin B (CTB) retrograde neural tracing, sciatic function index (SFI), angle of ankle, Hargreaves test and electrophysiological analysis. RESULTS: With AAV2-KLF7 injection, KLF7 expression increased in regenerated nerves, and amplitude, score of SFI, angle of ankle and FG-labelled spinal cord neurons were increased. We observed elevated CTB-labelled neurons in dorsal root ganglia (DRG), neurofilaments, P0 (peripheral myelin) and S100 and decreased latency period and withdrawal latencies in the Hargreaves test. The SFI was significantly correlated with amplitude and regenerated axon number. Tyrosine kinase A (TrkA) and B (TrkB) receptors were also increased in the DRG. CONCLUSIONS: Our findings suggest that KLF7 promoted peripheral nerve axonal regeneration, further supporting a role for KLF7 as a growth-promoting transcription factor in the injured nervous system.

Grafted bone marrow stromal cells: a contributor to glial repair after spinal cord injury.

In the CNS, astrocytes, oligodendrocytes and microglias are involved in not only development but also pathology such as spinal cord injury (SCI). Glial cells play dual roles (negative vs. positive effects) in these processes. After SCI, detrimental effects usually dominate and significantly retard functional recovery, and curbing these effects is critical for promoting neurological improvement. Bone marrow stromal cells (BMSCs) represent a new therapeutic approach for SCI by enabling improved sensory and motor functions in animal models. Although transdifferentiation to spinal neurons was poor, because of their pleiotropic nature, the protective effects of BMSCs are broad and are primarily mediated through modulation of transdifferentiation into host spinal glial components. Transplantation of BMSCs can positively alter the spinal microenvironment and enhance recovery. The objective of this review is to discuss these and other related mechanisms. Since BMSCs transplantation has been applied in other clinical fields, we hope to provide useful clues for the clinical application of BMSCs to treat the SCI in the near future.

Schwann cell transplantation and descending propriospinal regeneration after spinal cord injury.

After spinal cord injury (SCI), poor ability of damaged axons of the central nervous system (CNS) to regenerate causes very limited functional recovery. Schwann cells (SCs) have been widely explored as promising donors for transplantation to promote axonal regeneration in the CNS including the spinal cord. Compared with other CNS axonal pathways, injured propriospinal tracts display the strongest regenerative response to SC transplantation. Even without providing additional neurotrophic factors, propriospinal axons can grow into the SC environment which is rarely seen in supraspinal tracts. Propriospinal tract has been found to respond to several important neurotrophic factors secreted by SCs. Therefore, the SC is considered to be one of the most promising candidates for cell-based therapies for SCI. Since many reviews have already appeared on topics of SC transplantation in SCI repair, this review will focus particularly on the rationale of SC transplantation in mediating descending propriospinal axonal regeneration as well as optimizing such regeneration by using different combinatorial strategies. This article is part of a Special Issue entitled SI: Spinal cord injury.

Glucocorticoid Receptor ß Acts as a Co-activator of T-Cell Factor 4 and Enhances Glioma Cell Proliferation.

We previously reported that glucocorticoid receptor ß (GRß) regulates injury-mediated astrocyte activation and contributes to glioma pathogenesis via modulation of ß-catenin/T-cell factor/lymphoid enhancer factor (TCF/LEF) transcriptional activity. The aim of this study was to characterize the mechanism behind cross-talk between GRß and ß-catenin/TCF in the progression of glioma. Here, we reported that GRß knockdown reduced U118 and Shg44 glioma cell proliferation in vitro and in vivo. Mechanistically, we found that GRß knockdown decreased TCF/LEF transcriptional activity without affecting ß-catenin/TCF complex. Both GRα and GRß directly interact with TCF-4, while only GRß is required for sustaining TCF/LEF activity under hormone-free condition. GRß bound to the N-terminus domain of TCF-4 its influence on Wnt signaling required both ligand- and DNA-binding domains (LBD and DBD, respectively). GRß and TCF-4 interaction is enough to maintain the TCF/LEF activity at a high level in the absence of ß-catenin stabilization. Taken together, these results suggest a novel cross-talk between GRß and TCF-4 which regulates Wnt signaling and the proliferation in gliomas.

Cytosolic phospholipase A2 protein as a novel therapeutic target for spinal cord injury.

OBJECTIVE: The objective of this study was to investigate whether cytosolic phospholipase A2 (cPLA2 ), an important isoform of PLA2 that mediates the release of arachidonic acid, plays a role in the pathogenesis of spinal cord injury (SCI). METHODS: A combination of molecular, histological, immunohistochemical, and behavioral assessments were used to test whether blocking cPLA2 activation pharmacologically or genetically reduced cell death, protected spinal cord tissue, and improved behavioral recovery after a contusive SCI performed at the 10th thoracic level in adult mice. RESULTS: SCI significantly increased cPLA2 expression and activation. Activated cPLA2 was localized mainly in neurons and oligodendrocytes. Notably, the SCI-induced cPLA2 activation was mediated by the extracellular signal-regulated kinase signaling pathway. In vitro, activation of cPLA2 by ceramide-1-phosphate or A23187 induced spinal neuronal death, which was substantially reversed by arachidonyl trifluoromethyl ketone, a cPLA2 inhibitor. Remarkably, blocking cPLA2 pharmacologically at 30 minutes postinjury or genetically deleting cPLA2 in mice ameliorated motor deficits, and reduced cell loss and tissue damage after SCI. INTERPRETATION: cPLA2 may play a key role in the pathogenesis of SCI, at least in the C57BL/6 mouse, and as such could be an attractive therapeutic target for ameliorating secondary tissue damage and promoting recovery of function after SCI.

A novel growth-promoting pathway formed by GDNF-overexpressing Schwann cells promotes propriospinal axonal regeneration, synapse formation, and partial recovery of function after spinal cord injury.

Descending propriospinal neurons (DPSN) are known to establish functional relays for supraspinal signals, and they display a greater growth response after injury than do the long projecting axons. However, their regenerative response is still deficient due to their failure to depart from growth supportive cellular transplants back into the host spinal cord, which contains numerous impediments to axon growth. Here we report the construction of a continuous growth-promoting pathway in adult rats, formed by grafted Schwann cells overexpressing glial cell line-derived neurotrophic factor (GDNF). We demonstrate that such a growth-promoting pathway, extending from the axonal cut ends to the site of innervation in the distal spinal cord, promoted regeneration of DPSN axons through and beyond the lesion gap of a spinal cord hemisection. Within the distal host spinal cord, regenerated DPSN axons formed synapses with host neurons leading to the restoration of action potentials and partial recovery of function.

Cotransplantation of glial restricted precursor cells and Schwann cells promotes functional recovery after spinal cord injury.

Oligodendrocyte (OL) replacement can be a promising strategy for spinal cord injury (SCI) repair. However, the poor posttransplantation survival and inhibitory properties to axonal regeneration are two major challenges that limit their use as donor cells for repair of CNS injuries. Therefore, strategies aimed at enhancing the survival of grafted oligodendrocytes as well as reducing their inhibitory properties, such as the use of more permissive oligodendrocyte progenitor cells (OPCs), also called glial restricted precursor cells (GRPs), should be highly prioritized. Schwann cell (SC) transplantation is a promising translational strategy to promote axonal regeneration after CNS injuries, partly due to their expression and secretion of multiple growth-promoting factors. Whether grafted SCs have any effect on the biological properties of grafted GRPs remains unclear. Here we report that either SCs or SC-conditioned medium (SCM) promoted the survival, proliferation, and migration of GRPs in vitro. When GRPs and SCs were cografted into the normal or injured spinal cord, robust survival, proliferation, and migration of grafted GRPs were observed. Importantly, grafted GRPs differentiated into mature oligodendrocytes and formed new myelin on axons caudal to the injury. Finally, cografts of GRPs and SCs promoted recovery of function following SCI. We conclude that cotransplantation of GRPs and SCs, the only two kinds of myelin-forming cells in the nervous system, act complementarily and synergistically to promote greater anatomical and functional recovery after SCI than when either cell type is used alone.

The molecular events involved in oligodendrocyte precursor cell proliferation induced by the conditioned medium from b104 neuroblastoma cells.

The conditioned medium from B104 neuroblastoma cells (B104CM) induces proliferation of oligodendrocyte progenitor cells (OPCs) in vitro. However, the molecular events that occur during B104CM-induced proliferation of OPCs has not been well clarified. In the present study, using OPCs immunopanned from embryonic day 14 Sprague-Dawley rat spinal cords, we explored the activation of several signaling pathways and the expression of several important immediate early genes (IEGs) and cyclins in OPCs in response to B104CM. We found that B104CM can induce OPC proliferation through the activation of the extracellular signal-regulated kinases 1 and 2 (Erk1/2), but not PI3K or p38 MAPK signaling pathways in vitro. The IEGs involved in B104CM-induced OPC proliferation include c-fos, c-jun and Id2, but not c-myc, fyn, or p21. The cyclins D1, D2 and E are also involved in B104CM-stimulated proliferation of OPCs. The activation of Erk results in subsequent expression of IEGs (such as c-fos, c-jun and Id-2) and cyclins (including cyclin D1, D2 and E), which play key roles in cell cycle initiation and OPC proliferation. Collectively, these results suggest that the phosphorylation of Erk1/2 is an important molecular event during OPC proliferation induced by B104CM.

Pilose antler polypeptides promote chondrocyte proliferation via the tyrosine kinase signaling pathway.

BACKGROUND: Pilose antler polypeptides (PAP) have been reported to promote chondrocyte proliferation. However, the underlying mechanism remains unclear. The present study was to investigate the effects of PAP on the proliferation of chondrocytes and its underlying mechanism. METHODS: Chondrocytes isolated from the knee of Zealand white rabbits were cultured. The second generation chondrocytes were collected and identified using safranin-O staining. The chondrocytes were divided into the following 4 groups including serum-free, PAP, genistein (an inhibitor of tyrosine kinases), and PAP plus genistein group. Cell viability was analyzed using the MTT assay. The cell cycle distribution of the chondrocytes was analyzed by flow cytometry. The expression levels of cyclin A was detected using immunocytochemical staining. RESULTS: No significant difference was observed between serum-free and genistein group. Treatment of the cultures with PAP produced a significant dose-dependent increase in cell viability, the percentage proportion of chondrocytes in the S phase and Cyclin A expression as well. However, the promoting effect of PAP on chondrocyte proliferation were dose-dependently inhibited by genistein, whereas genistein alone had no effect on proliferation of isolated chondrocytes. CONCLUSIONS: The data demonstrate that PAP promotes chondrocyte proliferation with the increased cell number, percentage proportion of chondrocytes in S phase and expression of protein cyclin A via the TK signaling pathway.