Animal Venom Peptides as a Treasure Trove for New Therapeutics Against Neurodegenerative Disorders.

BACKGROUND: Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and cerebral ischemic stroke, impose enormous socio-economic burdens on both patients and health-care systems. However, drugs targeting these diseases remain unsatisfactory, and hence there is an urgent need for the development of novel and potent drug candidates. METHODS: Animal toxins exhibit rich diversity in both proteins and peptides, which play vital roles in biomedical drug development. As a molecular tool, animal toxin peptides have not only helped clarify many critical physiological processes but also led to the discovery of novel drugs and clinical therapeutics. RESULTS: Recently, toxin peptides identified from venomous animals, e.g. exenatide, ziconotide, Hi1a, and PcTx1 from spider venom, have been shown to block specific ion channels, alleviate inflammation, decrease protein aggregates, regulate glutamate and neurotransmitter levels, and increase neuroprotective factors. CONCLUSION: Thus, components of venom hold considerable capacity as drug candidates for the alleviation or reduction of neurodegeneration. This review highlights studies evaluating different animal toxins, especially peptides, as promising therapeutic tools for the treatment of different neurodegenerative diseases and disorders.

A Modular Assembly of Spinal Cord-Like Tissue Allows Targeted Tissue Repair in the Transected Spinal Cord.

Tissue engineering-based neural construction holds promise in providing organoids with defined differentiation and therapeutic potentials. Here, a bioengineered transplantable spinal cord-like tissue (SCLT) is assembled in vitro by simulating the white matter and gray matter composition of the spinal cord using neural stem cell-based tissue engineering technique. Whether the organoid would execute targeted repair in injured spinal cord is evaluated. The integrated SCLT, assembled by white matter-like tissue (WMLT) module and gray matter-like tissue (GMLT) module, shares architectural, phenotypic, and functional similarities to the adult rat spinal cord. Organotypic coculturing with the dorsal root ganglion or muscle cells shows that the SCLT embraces spinal cord organogenesis potentials to establish connections with the targets, respectively. Transplantation of the SCLT into the transected spinal cord results in a significant motor function recovery of the paralyzed hind limbs in rats. Additionally, targeted spinal cord tissue repair is achieved by the modular design of SCLT, as evidenced by an increased remyelination in the WMLT area and an enlarged innervation in the GMLT area. More importantly, the pro-regeneration milieu facilitates the formation of a neuronal relay by the donor neurons, allowing the conduction of descending and ascending neural inputs.

Recovery of paralyzed limb motor function in canine with complete spinal cord injury following implantation of MSC-derived neural network tissue.

We have reported previously that bone marrow mesenchymal stem cell (MSC)-derived neural network scaffold not only survived in the injury/graft site of spinal cord but also served as a "neuronal relay" that was capable of improving the limb motor function in a complete spinal cord injury (SCI) rat model. It remained to be explored whether such a strategy was effective for repairing the large spinal cord tissue loss as well as restoring motor function in larger animals. We have therefore extended in this study to construct a canine MSC-derived neural network tissue in vitro with the aim to evaluate its efficacy in treating adult beagle dog subjected to a complete transection of the spinal cord. The results showed that after co-culturing with neurotropin-3 overexpressing Schwann cells in a gelatin sponge scaffold for 14 days, TrkC overexpressing MSCs differentiated into neuron-like cells. In the latter, some cells appeared to make contacts with each other through synapse-like structures with trans-synaptic electrical activities. Remarkably, the SCI canines receiving the transplantation of the MSC-derived neural network tissue demonstrated a gradual restoration of paralyzed limb motor function, along with improved electrophysiological presentation when compared with the control group. Magnetic resonance imaging and diffusion tensor imaging showed that the canines receiving the MSC-derived neural network tissue exhibited robust nerve tract regeneration in the injury/graft site. Histological analysis showed that some of the MSC-derived neuron-like cells had survived in the injury/graft site up to 6.5 months. Implantation of MSC-derived neural network tissue significantly improved the microenvironment of the injury/graft site. It is noteworthy that a variable number of them had integrated with the regenerating corticospinal tract nerve fibers and 5-HT nerve fibers through formation of synapse-like contacts. The results suggest that the transplanted MSC-derived neural network tissue may serve as a structural and functional "neuronal relay" to restore the paralyzed limb motor function in the canine with complete SCI.

Microglial SMAD4 regulated by microRNA-146a promotes migration of microglia which support tumor progression in a glioma environment.

Glioma tumors constitute a significant portion of microglial cells, which are known to support tumor progression. The present study demonstrates that transforming growth factor-ß (TGFß) signaling pathway in microglia in a glioma environment is involved in tumor progression and pathogenesis. It has been shown that the TGFß level is elevated in higher grades of gliomas and its signaling pathway regulates tumor progression through phosphorylation of SMAD2 and SMAD3, which form a complex with SMAD4 to regulate target gene transcription. In an in vitro cell line-based model increased protein levels of pSMAD2/3, total SMAD2/3 and SMAD4 were observed in murine BV2 microglia cultured in glioma conditioned medium (GCM), indicative of the activated TGFß signaling pathway in microglia associated with glioma environment. Immunofluorescence labeling further revealed the expression of SMAD4 in microglial and non-microglial cells of human glioblastomas tissue in vivo. Functional analysis through shRNA-mediated stable knockdown of SMAD4 in microglia revealed the downregulation of the expression of matrix metalloproteinase 9 (MMP9), which has been shown to be involved in tumor progression and cell migration. Further, knockdown of SMAD4 in microglia decreased the migration of microglial cells towards GCM, indicating that SMAD4 promotes microglial migration in glioma environment. In addition, SMAD4 has been shown to be post-transcriptionally regulated by microRNA-146a, which was downregulated in microglia treated with GCM. Overexpression of miR-146a resulted in decreased expression of SMAD4 together with tumor supportive gene MMP9 in microglia, and subsequently suppressed microglial migration towards GCM, possibly through regulation of SMAD4. On the other hand, the cell viability assay revealed decreased viability of glioma cells when they were treated with conditioned medium derived from SMAD4 knockdown microglia or miR-146a overexpressed microglia as compared to glioma cells treated with the medium from control microglial cells. Taken together, the present study suggests that microglial SMAD4 which is epigenetically regulated by miR-146a promotes microglial migration in gliomas and glioma cell viability.

Herbal Compounds with Special Reference to Gastrodin as Potential Therapeutic Agents for Microglia Mediated Neuroinflammation.

BACKGROUND: Activated microglia play a pivotal role neurodegenerative diseases by producing a variety of proinflammatory mediators including tumor necrosis factor-alpha (TNF-α), interleukin- 1beta (IL-1ß) and nitric oxide (NO) that are toxic to neurons and oligodendrocytes. METHODS: In view of the above, suppression of microglia mediated neuroinflammation is deemed a therapeutic strategy for neurodegenerative diseases. Several potential Chinese herbal extracts have been reported to exert neuroprotective effects against neurodegenerative diseases targeting specifically at the activated microglia. In this connection, the phenolic glucoside gastrodin, a main constituent of the Chinese herbal medicine Gastrodia rhizoma, produced widely in the local community exhibits potential neuroprotective effects through suppression of neurotoxic proinflammatory mediators. RESULTS: Here, we first review the roles of activated microglia in different brain diseases. The effects of gastrodin on activated microglia are then considered. We have identified gastrodin as a putative therapeutic agent as it has been found to suppress microglial activation thus ameliorating neuroinflammation. More importantly, gastrodin downregulates the expression of renin angiotensin system (RAS) and production of proinflammatory mediators. Remarkably, gastrodin promotes Sirtuin 3 (Sirt3) up-regulation and nicotinamide adenine dinucleotide phosphate oxidase-2 (NOX-2) down-regulation after ischemichypoxia in activated microglia mediated by AT1 or AT2 receptors which are angiotensin II receptors subtypes, indicating a possible molecular link between RAS and Sirt3 survival genes. CONCLUSION: This review summarizes the beneficial effects of gastrodin acting on activated microglia along with other herbal compounds. Its efficacy in neuroprotection is consistent with some common herbal products in China.

Perineurium-like sheath derived from long-term surviving mesenchymal stem cells confers nerve protection to the injured spinal cord.

The functional multipotency enables mesenchymal stem cells (MSCs) promising translational potentials in treating spinal cord injury (SCI). Yet the fate of MSCs grafted into the injured spinal cord has not been fully elucidated even in preclinical studies, rendering concerns of their safety and genuine efficacy. Here we used a rat spinal cord transection model to evaluate the cell fate of allograft bone marrow derived MSCs. With the application of immunosuppressant, donor cells, delivered by biocompatible scaffold, survived up to 8 weeks post-grafting. Discernible tubes formed by MSCs were observed beginning 2 weeks after transplantation and they dominated the morphological features of implanted MSCs at 8 weeks post-grafting. The results of immunocytochemistry and transmission electron microscopy displayed the formation of perineurium-like sheath by donor cells, which, in a manner comparable to the perineurium in peripheral nerve, enwrapped host myelins and axons. The MSC-derived perineurium-like sheath secreted a group of trophic factors and permissive extracellular matrix, and served as a physical and chemical barrier to insulate the inner nerve fibers from ambient oxidative insults by the secretion of soluble antioxidant, superoxide dismutase-3 (SOD3). As a result, many intact regenerating axons were preserved in the injury/graft site following the forming of perineurium-like sheath. A parallel study utilizing a good manufacturing practice (GMP) grade human umbilical cord-derived MSCs or allogenic MSCs in an acute contusive/compressive SCI model exhibited a similar perineurium-like sheath formed by surviving donor cells in rat spinal cord at 3 weeks post-grafting. The present study for the first time provides an unambiguous morphological evidence of perineurium-like sheath formed by transplanted MSCs and a novel therapeutic mechanism of MSCs in treating SCI.

Transitory cystic cavities in the developing mammalian brain - normal or anomalous?

Transitory cavities associated with the ventricular system represent probably one of the most unique features in the developing mammalian brain. In rodents, the cavities exist transiently in the developing brain and do not appear to be associated with any pathological events. Among the various cavities, the pyramidal-shaped cavum septum pellucidum (CSP) located beneath the corpus callosum and between the lateral ventricles is most well defined. In addition to the CSP are the bilateral subependymal cysts that are consistently associated with the third and fourth ventricles as well as the aqueduct. The cavities/cysts contain a large number of amoeboid microglia expressing surface receptors and hydrolytic enzymes common to tissue macrophages. The significance of these cavities in the developing brain remains a conjecture. Firstly, the cavity walls are free of an apparent epithelial lining; hence, it is speculated that the cavities that appear to communicate with the widened neighboring interstitial tissue spaces may have resulted from physical traction due to the rapid growth of the perinatal brain. Secondly, the cavities contain prominent clusters of amoeboid microglia that may be involved in clearing the debris of degenerating axons and cells resulting from the early brain tissue remodeling. With the increase in brain tissue compactness following the beginning of myelination in the second postnatal week, all cavities are obliterated; concomitantly, the number of amoeboid microglia in them diminishes and all this might signal further maturation of the brain.

Glutamate Inhibits the Pro-Survival Effects of Insulin-Like Growth Factor-1 on Retinal Ganglion Cells in Hypoxic Neonatal Rat Retina.

Glutamate that accumulates in injured brain tissue has been shown to hinder the neuroprotection rendered by insulin-like growth factor-1 (IGF-1). However, its role in attenuating the neuroprotective effect of IGF-1 in the hypoxic retina is unknown and the current study was aimed at elucidating this. One-day-old Wistar rats were exposed to hypoxia for 2 h and the retinas were studied at 3 h to 14 days after exposure. Following hypoxia, the concentrations of glutamate and IGF-1 were significantly increased over control values in the immature retina and in cultured retinal ganglion cells (RGCs). In addition to IGF-1, the relative expression of insulin receptor substrate-1 (IRS1) phosphorylated at the tyrosine residue (p-IRS1tyr), phosphorylated protein kinase B (p-AKT) and phosphorylated protein kinase A (p-PKA), which are involved in IGF-1 signalling, was also studied in hypoxic retinas and in cultured RGCs. Glutamate-mediated inhibition of the IGF-1 pathway in hypoxic RGCs was evident with a reduced expression of p-IRS1tyr and p-AKT and an increased expression of p-PKA. However, the addition of exogenous IGF-1 reversed this. Glutamate enables the phosphorylation of IRS1 at the serine residue (p-IRS1ser) through a PKA-dependent pathway. The increased expression of p-IRS1ser and its increased association with IGF-1 receptors in hypoxic RGCs suggested a possible interference by glutamate with the IGF-1 pathway. Moreover, there was increased caspase-3/7 activity in hypoxic RGCs. These results suggest that glutamate, by blocking IGF-1-mediated neuroprotection, could cause the apoptosis of RGCs in the hypoxic neonatal retina.

Transplantation of tissue engineering neural network and formation of neuronal relay into the transected rat spinal cord.

Severe spinal cord injury (SCI) causes loss of neural connectivity and permanent functional deficits. Re-establishment of new neuronal relay circuits after SCI is therefore of paramount importance. The present study tested our hypothesis if co-culture of neurotrophin-3 (NT-3) gene-modified Schwann cells (SCs, NT-3-SCs) and TrkC (NT-3 receptor) gene-modified neural stem cells (NSCs, TrkC-NSCs) in a gelatin sponge scaffold could construct a tissue engineering neural network for re-establishing an anatomical neuronal relay after rat spinal cord transection. Eight weeks after transplantation, the neural network created a favorable microenvironment for axonal regeneration and for survival and synaptogenesis of NSC-derived neurons. Biotin conjugates of cholera toxin B subunit (b-CTB, a transneuronal tracer) was injected into the crushed sciatic nerve to label spinal cord neurons. Remarkably, not only ascending and descending nerve fibers, but also propriospinal neurons, made contacts with b-CTB positive NSC-derived neurons. Moreover, b-CTB positive NSC-derived neurons extended their axons making contacts with the motor neurons located in areas caudal to the injury/graft site of spinal cord. Further study showed that NT-3/TrkC interactions activated the PI3K/AKT/mTOR pathway and PI3K/AKT/CREB pathway affecting synaptogenesis of NSC-derived neurons. Together, our findings suggest that NT-3-mediated TrkC signaling plays an essential role in constructing a tissue engineering neural network thus representing a promising avenue for effective exogenous neuronal relay-based treatment for SCI.

Autocrine fibronectin from differentiating mesenchymal stem cells induces the neurite elongation in vitro and promotes nerve fiber regeneration in transected spinal cord injury.

Extracellular matrix (ECM) expression is temporally and spatially regulated during the development of stem cells. We reported previously that fibronectin (FN) secreted by bone marrow mesenchymal stem cells (MSCs) was deposited on the surface of gelatin sponge (GS) soon after culture. In this study, we aimed to assess the function of accumulated FN on neuronal differentiating MSCs as induced by Schwann cells (SCs) in three dimensional transwell co-culture system. The expression pattern and amount of FN of differentiating MSCs was examined by immunofluorescence, Western blot and immunoelectron microscopy. The results showed that FN accumulated inside GS scaffold, although its mRNA expression in MSCs was progressively decreased during neural induction. MSC-derived neuron-like cells showed spindle-shaped cell body and long extending processes on FN-decorated scaffold surface. However, after blocking of FN function by application of monoclonal antibodies, neuron-like cells showed flattened cell body with short and thick neurites, together with decreased expression of integrin ß1. In vivo transplantation study revealed that autocrine FN significantly facilitated endogenous nerve fiber regeneration in spinal cord transection model. Taken together, the present results showed that FN secreted by MSCs in the early stage accumulated on the GS scaffold and promoted the neurite elongation of neuronal differentiating MSCs as well as nerve fiber regeneration after spinal cord injury. This suggests that autocrine FN has a dynamic influence on MSCs in a three dimensional culture system and its potential application for treatment of traumatic spinal cord injury. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1902-1911, 2016.

Cell Transplantation and Neuroengineering Approach for Spinal Cord Injury Treatment: A Summary of Current Laboratory Findings and Review of Literature.

Spinal cord injury (SCI) can cause severe traumatic injury to the central nervous system (CNS). Current therapeutic effects achieved for SCI in clinical medicine show that there is still a long way to go to reach the desired goal of full or significant functional recovery. In basic medical research, however, cell transplantation, gene therapy, application of cytokines, and biomaterial scaffolds have been widely used and investigated as treatments for SCI. All of these strategies when used separately would help rebuild, to some extent, the neural circuits in the lesion area of the spinal cord. In light of this, it is generally accepted that a combined treatment may be a more effective strategy. This review focuses primarily on our recent series of work on transplantation of Schwann cells and adult stem cells, and transplantation of stem cell-derived neural network scaffolds with functional synapses. Arising from this, an artificial neural network (an exogenous neuronal relay) has been designed and fabricated by us-a biomaterial scaffold implanted with Schwann cells modified by the neurotrophin-3 (NT-3) gene and adult stem cells modified with the TrkC (receptor of NT-3) gene. More importantly, experimental evidence suggests that the novel artificial network can integrate with the host tissue and serve as an exogenous neuronal relay for signal transfer and functional improvement of SCI.

Graft of the NT-3 persistent delivery gelatin sponge scaffold promotes axon regeneration, attenuates inflammation, and induces cell migration in rat and canine with spinal cord injury.

Persistent neurotrophic factor delivery is crucial to create a microenvironment for cell survival and nerve regeneration in spinal cord injury (SCI). This study aimed to develop a NT-3/fibroin coated gelatin sponge scaffold (NF-GS) as a novel controlled artificial release therapy for SCI. In vitro, bone marrow-derived mesenchymal stem cells (MSCs) were planted into the NF-GS and release test showed that NF-GS was capable to generate a sustainable NT-3 release up to 28 days. MSCs in NF-GS had high cell activity with excellent cell distribution and phenotype. Then, the NF-GS was transplanted into the injury site of spinal cord of rat and canine in vivo, which exhibited strong biocompatibility during post-transplantation period. Four weeks following transplantation, the concentration of NT-3 was much higher than that in control groups. Cavity areas in the injury/graft site were significantly reduced due to tissue regeneration and axonal extensions associated with myelin sheath through the glial scar into the NF-GS. Additionally, the NF-GS decreased the inflammation by reducing the CD68 positive cells and TNF-α. A striking feature was the occurrence of some cells and myelin-like structure that appeared to traverse the NF-GS. The present results demonstrate that the NF-GS has the property to control the release of NT-3 from the NT-3/fibroin complex thus facilitating regeneration of injured spinal cord.

Hypoxia-Induced Iron Accumulation in Oligodendrocytes Mediates Apoptosis by Eliciting Endoplasmic Reticulum Stress.

This study was aimed at evaluating the role of increased iron accumulation in oligodendrocytes and its role in their apoptosis in the periventricular white matter damage (PWMD) following a hypoxic injury to the neonatal brain. In response to hypoxia, in the PWM, there was increased expression of proteins involved in iron acquisition, such as iron regulatory proteins (IRP1, IRP2) and transferrin receptor in oligodendrocytes. Consistent with this, following a hypoxic exposure, there was increased accumulation of iron in primary cultured oligodendrocytes. The increased concentration of iron within hypoxic oligodendrocytes was found to elicit ryanodine receptor (RyR) expression, and the expression of endoplasmic reticulum (ER) stress markers such as binding-immunoglobulin protein (BiP) and inositol-requiring enzyme (IRE)-1α. Associated with ER stress, there was reduced adenosine triphosphate (ATP) levels within hypoxic oligodendrocytes. However, treatment with deferoxamine reduced the increased expression of RyR, BiP, and IRE-1α and increased ATP levels in hypoxic oligodendrocytes. Parallel to ER stress there was enhanced reactive oxygen species production within mitochondria of hypoxic oligodendrocytes, which was attenuated when these cells were treated with deferoxamine. At the ultrastructural level, hypoxic oligodendrocytes frequently showed dilated ER and disrupted mitochondria, which became less evident in those treated with deferoxamine. Associated with these subcellular changes, the apoptosis of hypoxic oligodendrocytes was evident with an increase in p53 and caspase-3 expression, which was attenuated when these cells were treated with deferoxamine. Thus, the present study emphasizes that the excess iron accumulated within oligodendrocytes in hypoxic PWM could result in their death by eliciting ER stress and mitochondrial disruption.

Donor mesenchymal stem cell-derived neural-like cells transdifferentiate into myelin-forming cells and promote axon regeneration in rat spinal cord transection.

INTRODUCTION: Severe spinal cord injury often causes temporary or permanent damages in strength, sensation, or autonomic functions below the site of the injury. So far, there is still no effective treatment for spinal cord injury. Mesenchymal stem cells (MSCs) have been used to repair injured spinal cord as an effective strategy. However, the low neural differentiation frequency of MSCs has limited its application. The present study attempted to explore whether the grafted MSC-derived neural-like cells in a gelatin sponge (GS) scaffold could maintain neural features or transdifferentiate into myelin-forming cells in the transected spinal cord. METHODS: We constructed an engineered tissue by co-seeding of MSCs with genetically enhanced expression of neurotrophin-3 (NT-3) and its high-affinity receptor tropomyosin receptor kinase C (TrkC) separately into a three-dimensional GS scaffold to promote the MSCs differentiating into neural-like cells and transplanted it into the gap of a completely transected rat spinal cord. The rats received extensive post-operation care, including cyclosporin A administrated once daily for 2 months. RESULTS: MSCs modified genetically could differentiate into neural-like cells in the MN + MT (NT-3-MSCs + TrKC-MSCs) group 14 days after culture in the GS scaffold. However, after the MSC-derived neural-like cells were transplanted into the injury site of spinal cord, some of them appeared to lose the neural phenotypes and instead transdifferentiated into myelin-forming cells at 8 weeks. In the latter, the MSC-derived myelin-forming cells established myelin sheaths associated with the host regenerating axons. And the injured host neurons were rescued, and axon regeneration was induced by grafted MSCs modified genetically. In addition, the cortical motor evoked potential and hindlimb locomotion were significantly ameliorated in the rat spinal cord transected in the MN + MT group compared with the GS and MSC groups. CONCLUSION: Grafted MSC-derived neural-like cells in the GS scaffold can transdifferentiate into myelin-forming cells in the completely transected rat spinal cord.

Integration of donor mesenchymal stem cell-derived neuron-like cells into host neural network after rat spinal cord transection.

Functional deficits following spinal cord injury (SCI) primarily attribute to loss of neural connectivity. We therefore tested if novel tissue engineering approaches could enable neural network repair that facilitates functional recovery after spinal cord transection (SCT). Rat bone marrow-derived mesenchymal stem cells (MSCs), genetically engineered to overexpress TrkC, receptor of neurotrophin-3 (NT-3), were pre-differentiated into cells carrying neuronal features via co-culture with NT-3 overproducing Schwann cells in 3-dimensional gelatin sponge (GS) scaffold for 14 days in vitro. Intra-GS formation of MSC assemblies emulating neural network (MSC-GS) were verified morphologically via electron microscopy (EM) and functionally by whole-cell patch clamp recording of spontaneous post-synaptic currents. The differentiated MSCs still partially maintained prototypic property with the expression of some mesodermal cytokines. MSC-GS or GS was then grafted acutely into a 2 mm-wide transection gap in the T9-T10 spinal cord segments of adult rats. Eight weeks later, hindlimb function of the MSC-GS-treated SCT rats was significantly improved relative to controls receiving the GS or lesion only as indicated by BBB score. The MSC-GS transplantation also significantly recovered cortical motor evoked potential (CMEP). Histologically, MSC-derived neuron-like cells maintained their synapse-like structures in vivo; they additionally formed similar connections with host neurites (i.e., mostly serotonergic fibers plus a few corticospinal axons; validated by double-labeled immuno-EM). Moreover, motor cortex electrical stimulation triggered c-fos expression in the grafted and lumbar spinal cord cells of the treated rats only. Our data suggest that MSC-derived neuron-like cells resulting from NT-3-TrkC-induced differentiation can partially integrate into transected spinal cord and this strategy should be further investigated for reconstructing disrupted neural circuits.

Combination of electroacupuncture and grafted mesenchymal stem cells overexpressing TrkC improves remyelination and function in demyelinated spinal cord of rats.

This study attempted to graft neurotrophin-3 (NT-3) receptor (TrkC) gene modified mesenchymal stem cells (TrkC-MSCs) into the demyelinated spinal cord and to investigate whether electroacupuncture (EA) treatment could promote NT-3 secretion in the demyelinated spinal cord as well as further enhance grafted TrkC-MSCs to differentiate into oligodendrocytes, remyelination and functional recovery. Ethidium bromide (EB) was microinjected into the spinal cord of rats at T10 to establish a demyelinated model. Six groups of animals were prepared for the experiment: the sham, PBS, MSCs, MSCs+EA, TrkC-MSCs and TrkC-MSCs+EA groups. The results showed that TrkC-MSCs graft combined with EA treatment (TrkC-MSCs+EA group) significantly increased the number of OPCs and oligodendrocyte-like cells differentiated from MSCs. Immunoelectron microscopy showed that the oligodendrocyte-like cells differentiated from TrkC-MSCs formed myelin sheaths. Immunofluorescence histochemistry and Western blot analysis indicated that TrkC-MSCs+EA treatment could promote the myelin basic protein (MBP) expression and Kv1.2 arrangement trending towards the normal level. Furthermore, behavioural test and cortical motor evoked potentials detection demonstrated a significant functional recovery in the TrkC-MSCs+EA group. In conclusion, our results suggest that EA treatment can increase NT-3 expression, promote oligodendrocyte-like cell differentiation from TrkC-MSCs, remyelination and functional improvement of demyelinated spinal cord.

Scutellarin regulates the Notch pathway and affects the migration and morphological transformation of activated microglia in experimentally induced cerebral ischemia in rats and in activated BV-2 microglia.

BACKGROUND: Activated microglial cells release an excess of inflammatory mediators after an ischemic stroke. We reported previously that scutellarin effectively suppressed the inflammatory response induced by activated microglia in rats subjected to middle cerebral artery occlusion (MCAO); however, the mechanism via which scutellarin exerts its effects on microglial activation has not been explored. This study aimed to elucidate if scutellarin can regulate the Notch pathway that is linked to microglia activation in MCAO rat, and in lipopolysaccharide (LPS)-induced BV-2 microglia. Along with this, we also investigated some characteristic behavioral responses of activated microglia. METHODS: Expression of various members of the Notch pathway, including Notch-1, intracellular Notch receptor domain (NICD), recombining binding protein suppressor of hairless (RBP-JK) and transcription factor hairy and enhancer of split-1 (Hes-1) in activated microglia was assessed by immunofluorescence staining and western blot after experimental MCAO and in vitro LPS activation. The effect of scutellarin on migration of microglia was determined by the transwell chamber assay as well as expression of monocyte chemoattractant protein-1 (MCP-1). The morphological change of microglia induced by scutellarin was detected by F-actin staining and electron microscopy. RESULTS: Scutellarin markedly attenuated the expression of NF-κB, Notch-1, NICD, RBP-JK and Hes-1 both in vivo and in activated microglia. It decreased the expression of MCP-1 and microglial migration, but increased the ability of microglia adhesion. Scutellarin also altered the phenotype of microglia by causing rearrangement or reorganization of its cytoskeleton. CONCLUSIONS: The results suggest that scutellarin regulates the activation of microglia via the Notch pathway and concurrently induces morphological and functional changes in activated microglia.

Electroacupuncture Promotes the Differentiation of Transplanted Bone Marrow Mesenchymal Stem Cells Preinduced With Neurotrophin-3 and Retinoic Acid Into Oligodendrocyte-Like Cells in Demyelinated Spinal Cord of Rats.

Transplantation of bone marrow mesenchymal stem cells (MSCs) promotes functional recovery in multiple sclerosis (MS) patients and in a murine model of MS. However, there is only a modicum of information on differentiation of grafted MSCs into oligodendrocyte-like cells in MS. The purpose of this study was to transplant neurotrophin-3 (NT-3) and retinoic acid (RA) preinduced MSCs (NR-MSCs) into a demyelinated spinal cord induced by ethidium bromide and to investigate whether EA treatment could promote NT-3 secretion in the demyelinated spinal cord. We also sought to determine whether increased NT-3 could further enhance NR-MSCs overexpressing the tyrosine receptor kinase C (TrkC) to differentiate into more oligodendrocyte-like cells, resulting in increased remyelination and nerve conduction in the spinal cord. Our results showed that NT-3 and RA increased transcription of TrkC mRNA in cultured MSCs. EA increased NT-3 levels and promoted differentiation of oligodendrocyte-like cells from grafted NR-MSCs in the demyelinated spinal cord. There was evidence of myelin formation by grafted NR-MSCs. In addition, NR-MSC transplantation combined with EA treatment (the NR-MSCs + EA group) reduced demyelination and promoted remyelination. Furthermore, the conduction of cortical motor-evoked potentials has improved compared to controls. Together, our data suggest that preinduced MSC transplantation combined with EA treatment not only increased MSC differentiation into oligodendrocyte-like cells forming myelin sheaths, but also promoted remyelination and functional improvement of nerve conduction in the demyelinated spinal cord.

Anti-inflammatory effects of Edaravone and Scutellarin in activated microglia in experimentally induced ischemia injury in rats and in BV-2 microglia.

BACKGROUND: In response to cerebral ischemia, activated microglia release excessive inflammatory mediators which contribute to neuronal damage. Therefore, inhibition of microglial over-activation could be a therapeutic strategy to alleviate various microglia-mediated neuroinflammation. This study was aimed to elucidate the anti-inflammatory effects of Scutellarin and Edaravone given either singly, or in combination in activated microglia in rats subjected to middle cerebral artery occlusion (MCAO), and in lipopolysaccharide (LPS)-induced BV-2 microglia. Expression of proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and inducible nitric oxide synthase (iNOS) was assessed by immunofluorescence staining and Western blot. Reactive oxygen species (ROS) and nitric oxide (NO) levels were determined by flow cytometry and fluorescence microscopy, respectively. RESULTS: In vivo, both Edaravone and Scutellarin markedly reduced the infarct cerebral tissue area with the latter drug being more effective with the dosage used; furthermore, when used in combination the reduction was more substantial. Remarkably, a greater diminution in distribution of activated microglia was observed with the combined drug treatment which also attenuated the immunoexpression of TNF-α, IL-1ß and iNOS to a greater extent as compared to the drugs given separately. In vitro, both drugs suppressed upregulated expression of inflammatory cytokines, iNOS, NO and ROS in LPS-induced BV-2 cells. Furthermore, Edaravone and Scutellarin in combination cumulatively diminished the expression levels of the inflammatory mediators being most pronounced for TNF-α as evidenced by Western blot. CONCLUSION: The results suggest that Edaravone and Scutellarin effectively suppressed the inflammatory responses in activated microglia, with Scutellarin being more efficacious within the dosage range used. Moreover, when both drugs were used in combination, the infarct tissue area was reduced more extensively; also, microglia-mediated inflammatory mediators notably TNF-α expression was decreased cumulatively.

Therapeutic implications of melatonin in cerebral edema.

Cerebral edema/brain edema refers to the accumulation of fluid in the brain and is one of the fatal conditions that require immediate medical attention. Cerebral edema develops as a consequence of cerebral trauma, cerebral infarction, hemorrhages, abscess, tumor, hypoxia, and other toxic or metabolic factors. Based on the causative factors cerebral edema is differentiated into cytotoxic cerebral edema, vasogenic cerebral edema, osmotic and interstitial cerebral edema. Treatment of cerebral edema depends on timely diagnosis and medical assistance. Pragmatic treatment strategies such as antihypertensive medications, nonsteroidal anti-inflammatory drugs, barbiturates, steroids, glutamate and N-methyl-D-aspartate receptor antagonists and trometamol are used in clinical practice. Although the above mentioned treatment approaches are being used, owing to the complexity of the mechanisms involved in cerebral edema, a single therapeutic strategy which could ameliorate cerebral edema is yet to be identified. However, recent experimental studies have suggested that melatonin, a neurohormone produced by the pineal gland, could be an effective alternative for treating cerebral edema. In animal models of stroke, melatonin was not only shown to reduce cerebral edema but also preserved the blood brain barrier. Melatonin's beneficial effects were attributed to its properties, such as being a potent anti-oxidant, and its ability to cross the blood brain barrier within minutes after its administration. This review summarizes the beneficial effects of melatonin when used for treating cerebral edema.