MAP2-positive Cells Research Articles

Effects of salvianolate lyophilized injection on neural functional recovery and the expression of microtubule associated protein-2 after focal cerebral ischemia-reperfusion injury in diabetic rats

Objective To investigate the effects of salvianolate lyophilized injection(SLI) on neural functional recovery and the expression of microtubule associated protein-2(MAP2) after focal cerebral ischemia-reperfusion injury in the diabetic rats. Methods Diabetes model was made by intraperitoneal injection of streptozotocin (STZ) and cerebral ischemia-reperfusion model was developed by Longa suture occluded method in the middle cerebral artery of diabetic rats. The rats were randomly divided into five groups: sham operation group, model group, SLI (21.0 mg.kg-1, 10.5 mg.kg-1) treatment groups, and edaravone (6 mg.kg-1) treatment group.3 hours after ischemia, rats were respectively given normal saline or drugs followed by the injection once a day for 14 days and the neurological impairment was assessed. 2 h after the last injection, the rats were decapitated and the brains were collected. The expression of MAP2 protein and mRNA in the bilateral hippocampal ischemia and infarcted area was detected with immunohistochemistry and RT-PCR. Results Severe neurological dysfunction was found in diabetic rats that had been subjected to cerebral ischemic injury(1.850±0.457). A significant improvement on neurological function was found in the SLI treatment groups(1.581±0.314, 1.345±0.425) compared with model group(P<0.01, P<0.05=. Moreover, the expression of MAP2 in ischemia bilateral hippocampal CA1 and penumbra was represented by the average optical density value respectively (0.743±0.250, 0.561±0.224 ). In the hippocampal CA1 region, the number of MAP2-positive cells(0.781±0.420, 0.851±0.136) in the treatment group showed significant increase than those in model group (P<0.01, P<0.05=. In the ischemic penumbra region, the number of MAP2-positive cells (0.753±0.235, 1.203±0.326 ) in the treatment group showed significant increase than those in model group (P<0.01, P<0.05=. Conclusion The SLI can promote the post-injury neurocognitive function in diabetic rats. The increase of MAP2 expression may be involved in the mechanisms. Key words: Diabetes; Cerebral ischemia-reperfusion injury; Salvianolate lyophilized injection; Microtubule associated protein-2

PP2 - Modulation of oxidative stress in human hippocampal progenitor cells: a model to study underlying mechanisms of depression

Background: Recent findings suggest that oxidative stress (OS) plays an important role in the pathophysiology of depression. Excessive reactive oxygen species (ROS) alter neural cytoarchitecture and function and could contribute to lower neuroplasticity and reduced neurogenesis, both of which have been linked to lower mood. This study aimed to explore the effects of OS on human hippocampal progenitor cells capable of neurogenesis. Methods: We induced OS in human hippocampal progenitor cells by treatment with tert-butylhydroperoxide (T-BHP) in different concentrations. Cell viability was assessed by the MTS assay. Level of malondialdehyde (MDA) was measured in cells supernatant using the TBARS assay. Activation of Nf-kB and Nrf2 transcription factors were followed by immunostaining and TransAM kit respectively. After differentiation cells were labeled with DCX and MAP2 – markers of young and mature neurons and assessed using CellInsight platform. Results: At 100 μM, 200 μM and 500 μM, T-BHP caused a reduction of cell viability for 23%, 54%, and 74% respectively, compared with vehicle, with a concomitant increase in levels of MDA. At 50 μM and 100 μM of T-BHP, Nf-κB positive cells increased by 16% and 17% respectively, while Nrf2 dose-dependently translocated to the nucleus. At 1 μM, 10 μM and 25 μM, T-BHP increased MAP2 and DCX positive cells by 34%, 33% and 24% for MAP2 and 38% and 30% for at 1 μM and 10 μM for DCX. Conclusions: Our results show that cell damage and cell death of human hippocampal progenitor cells caused by T-BHP occur in a dose-dependent manner. We found activation of transcription factors Nf-κB and Nrf2 in response to OS stimuli. Our results also indicate higher levels of neurogenesis in cells treated with low doses of T-BHP. Thus, we conclude that ROS serve as secondary messengers and facilitate various signaling pathways regulating fundamental neurobiological processes known to be affected in depression.

In Vitro Differentiation of Human Neural Progenitor Cells Into Striatal GABAergic Neurons.

: Huntington's disease (HD) results from a CAG repeat expansion in the gene encoding the huntingtin protein. This inherited disorder is characterized by progressive neurodegeneration. In particular, HD progression involves the loss of striatal projection neurons. The limited availability of reliable sources of human striatal projection neurons currently hampers our understanding of HD mechanisms and hinders the development of novel HD treatments. In this paper, we described two- and three-step methods for differentiating human neural progenitor cells toward striatal projection neurons. In the two-step differentiation protocol, 90%, 54%, and 6% of MAP2-positive cells were immunopositive for GABA, calbindin (CALB1), and DARPP-32/PPP1R1B, respectively. In the three-step differentiation protocol, 96%, 84%, and 21% of MAP2-positive cells were immunopositive for GABA, calbindin, and DARPP-32/PPP1R1B, respectively. In line with a striatal projection neuron phenotype, cells differentiated with our protocols displayed significantly increased expression of MAP2, CALB1, DARPP-32/PPP1R1B, ARPP21, and CTIP2. Application of glutamate receptor agonists induced calcium influx; accordingly, the cells also expressed various ionotropic glutamate receptor subunits. Differentiated cells also released GABA on stimulation. We suggest that our three-step differentiation protocol presents a reliable and simplified method for the generation of striatal projection neurons, yielding a critical resource for neuronal physiology and neurodegenerative disorder studies. The earliest changes in the neurodegenerative disorder Huntington's disease affect a specific type of brain neurons, the so-called medium spiny neurons of the striatum. In this study, two protocols were developed for the differentiation of neural progenitor cells into striatal medium spiny neurons, and the differentiated neurons were extensively characterized. The data indicate that the three-step differentiation protocol presents a reliable and simplified method for the generation of striatal medium spiny neurons. The generated striatal medium spiny neurons could represent a critical resource for the study of neurodegenerative disorders, a model system for drug discovery, and a step toward cell-based regeneration therapies.

Neurogenic potential of spinal cord organotypic culture

There are several neurogenic niches in the adult mammalian central nervous system. In the central nervous system, neural stem cells (NSC) localize not only to the periventricular area, but are also diffusely distributed in the parenchyma. Here, we assessed neurogenic potential of organotypic cultures prepared from adult mouse spinal cord. Slices were placed on Millipore inserts for organotypic culture and incubated in neurobasal media supplemented with B27 and N2 for up to 9 weeks. After 3–4 weeks, the cell’s aggregates formed in the slices. The aggregate’s cells were BrdU-uptake, nestin and alkaline phosphatase positive. At the later stage of incubation, we observed Oct3/4 in the inner mass of the neurospheres as well as expression of Dppa1, which is an Oct-4 downstream target gene and a marker for pluripotency. To check differentiation, the formed neurospheres were isolated and cultured for several days in differentiation media. The obtained data demonstrated the cells from isolated neurospheres differentiate into astrocytes and MAP2-positive neurons. Immunostaining for HB9 and Lim2 revealed subsequent differentiation of MAP2-positive cells into motor neurons and interneurons, respectively. We hypothesized neuronal loss and/or long-term culturing of spinal cord slices may trigger a reset of the internal cell program and promote proliferation and further differentiation of NSC.

Expression of a novel serine/threonine kinase gene, Ulk4, in neural progenitors during Xenopus laevis forebrain development

We have analyzed the expression pattern of a novel serine/threonine kinase gene Ulk4 during forebrain development in Xenopus laevis. To this aim, we firstly cloned a Ulk4 cDNA fragment from X.laevis and generated a RNA probe that was used for its detection by in situ hybridization. Throughout development xUlk4 expression was detected along the ventricular (vz) and subventricular zones (svz) of all forebrain regions, with the exception of the vz of the striatum. In the adult, xUlk4 was also mainly located in the vz, with some xUlk4 expressing cells reaching the svz/mantle zone (mz). This xUlk4 expression was especially remarkable in forebrain regions involving the homeostatic control of the brain such as the preoptic region, the hypothalamic territory and some neurosecretory circumventricular organs (CVOs). We further combined in situ hybridization for xUlk4 with immunohistochemistry for the neural progenitor cell marker SOX3, the radial glial marker brain lipid-binding protein (BLBP), neuronal markers MAP2 and doublecortin (DCX) and the specific neuronal marker tyrosine hydroxylase (TH). xUlk4 was co-expressed with the neural stem/progenitor cell marker SOX3 in the vz of all the forebrain regions throughout development and in the adult, and this co-expression was also especially evident in the svz of the hypothalamic region. xUlk4 was also expressed in the radial glia along the whole brain. We have also found minor expression of xUlk4 in some DCX- or MAP2-positive cells but not in TH-positive neurons. These findings suggest that Ulk4 may play roles in neural stem/progenitor cells during neurogenesis both in development and in the adulthood, in migrating cells as well as in cells committed to neuronal fate in Xenopus. Moreover, the results obtained in this study argue for an involvement of Ulk4 in the control of the neuroendocrine homeostatic functions in the brain.

Graft of the gelatin sponge scaffold containing genetically-modified neural stem cells promotes cell differentiation, axon regeneration, and functional recovery in rat with spinal cord transection.

Biological materials combined with genetically-modified neural stem cells (NSCs) are candidate therapy targeting spinal cord injury (SCI). Based on our previous studies, here we performed gelatin sponge (GS) scaffold seeded with neurotrophin-3 (NT-3) and its receptor TrkC gene modifying NSCs for repairing SCI. Eight weeks later, compared with other groups, neurofilament-200 and 5-hydroxytryptamine positive nerve fibers were more in the injury site of the N+T-NSCs group. Immunofluorescence staining showed the grafted NSCs could differentiate into microtubule associated protein (Map2), postsynaptic density (PSD95), and mouse oligodendrocyte special protein (MOSP) positive cells. The percentage of the Map2, PSD95, and MOSP positive cells in the N+T-NSCs group was higher than the other groups. Immuno-electron microscopy showed the grafted NSCs making contact with each other in the injury site. Behavioral analysis indicated the recovery of hindlimbs locomotion was better in the groups receiving cell transplant, the best recovery was found in the N+T-NSCs group. Electrophysiology revealed the amplitude of cortical motor evoked potentials was increased significantly in the N+T-NSCs group, but the latency remained long. These findings suggest the GS scaffold containing genetically-modified NSCs may bridge the injury site, promote axon regeneration and partial functional recovery in SCI rats.

Effect of human umbilical cord blood mesenchymal stem cell transplantation on neuronal metabolites in ischemic rabbits

BackgroundBecause there is little research on the effects of transplanted stem cells on neuronal metabolites in infarct areas, we transplanted human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) into cerebral ischemic rabbits and examined the neuronal metabolites.ResultsRabbits (n = 40) were equally divided into sham, middle cerebral artery occlusion (MCAO), hUCB-MSC, and saline groups. The rabbit ischemic model was established by MCAO. The effects of hUCB-MSC transplantation were assessed by proton magnetic resonance spectroscopy (1H-MRS), neurological severity scores (NSSs), infarct area volume, neuronal density, and optical density (OD) of microtubule-associated protein 2 (MAP2)-positive cells. We also evaluated complete blood cell counts(CBCs) and serum biochemical parameters. NSSs in the hUCB-MSC group at 7 and 14 days after reperfusion were lower than in MCAO and saline groups (p < 0.05). Compared with MCAO and saline groups at 2 weeks after MCAO, the infarction volume in the hUCB-MSC group had decreased remarkably (p < 0.05). Significant neuronal metabolic changes occurred in the infarct area at 24 h and 2 weeks after MCAO. 1H-MRS revealed an elevation in the lactate (Lac)/creatine including phosphocreatine (Cr) ratio and a decrease in the N-acetylaspartate (NAA)/Cr and choline-containing phospholipids (Cho)/Cr ratios at 24 h after MCAO in the MCAO group (p < 0.01). Compared with saline and MCAO groups at 24 h and 2 weeks after MCAO, NAA/Cr and Cho/Cr ratios had increased significantly, whereas the Lac/Cr ratio had decreased significantly in the hUCB-MSC group (p < 0.01). Neuronal density and OD of MAP2-positive cells in the MCAO group were significantly lower than those in the sham group, whereas the neuronal density and OD of MAP2-positive cells in the hUCB-MSC group were higher than those in MCAO and saline groups (p < 0.05). CBCs and biochemical parameters were unchanged in the MCAO group at 24 h and 2 weeks after hUCB-MSC transplantation.ConclusionsTransplanted hUCB-MSCs might ameliorate ischemic damage by influencing neuronal metabolites in the infarct area, providing additional evidence for neuroprotection by stem cells. No significant changes were observed in CBCs or serum biochemical parameters, suggesting that intravenous infusion of hUCB-MSCs is safe for rabbits in the short-term.

Analysis of the antiepileptic, ethosuximide impacts on neurogenesis of rat forebrain stem cells

Specific GABAergic interneurons in the hilus are lost in animal models with temporal-lobe epilepsy (TLE). Some preclinical evidence has indicated that GABAergic cells may provide relief from seizures in these models. This study was aimed to examine the ability of ethosuximide, an anticonvulsant drug, to promote neurogenesis in 3-day-old rat forebrain cortex stem cells. Most of the cells were found to be nestin-positive undifferentiated neural stem cells prior to their exposure to ethosuximide. It was noted that the number and percentage of tubulin β-III immunopositive neurons were increased after 6 days treatment with ethosuximide. Upon bFGF withdrawal, exposure to ethosuximide differentiated the stem cells to MAP2 positive neural cells (7.18 ± 0.43, 21.766 ± 0.55 and 41.57 ± 0.5 for control, 0.1 and 1 μM, respectively). GABA immunofluorescence images illustrated that ethosuximide increased GABAergic neurons (7.19 ± 0.32, 23.23 ± 0.55, and 46.30 ± 0.44 for control, 0.1 and 1 μM, respectively). Additionally, BrdU immunofluorescence assay showed that ethosuximide-enhanced nucleus proliferation in the neuronal stem cells. Therefore, the results of this study suggest that ethosuximide may compensate damage caused by seizure attacks and possibly other neuronal loss disorders.

Traumatic Brain Injury Precipitates Cognitive Impairment and Extracellular Aβ Aggregation in Alzheimer's Disease Transgenic Mice

Traumatic brain injury (TBI) has become a signature wound of the wars in Iraq and Afghanistan. Many American soldiers, even those undiagnosed but likely suffering from mild TBI, display Alzheimer's disease (AD)-like cognitive impairments, suggesting a pathological overlap between TBI and AD. This study examined the cognitive and neurohistological effects of TBI in presymptomatic APP/PS1 AD-transgenic mice. AD mice and non-transgenic (NT) mice received an experimental TBI on the right parietal cortex using the controlled cortical impact model. Animals were trained in a water maze task for spatial memory before TBI, and then reevaluated in the same task at two and six weeks post-TBI. The results showed that AD mice with TBI made significantly more errors in the task than AD mice without TBI and NT mice regardless of TBI. A separate group of AD mice and NT mice were evaluated neurohistologically at six weeks after TBI. The number of extracellular beta-amyloid (Aβ)-deposits significantly increased by at least one fold in the cortex of AD mice that received TBI compared to the NT mice that received TBI or the AD and NT mice that underwent sham surgery. A significant decrease in MAP2 positive cells, indicating neuronal loss, was observed in the cortex of both the AD and NT mice that received TBI compared to the AD and NT mice subjected to sham surgery. Similar changes in extracellular Aβ deposits and MAP2 positive cells were also seen in the hippocampus. These results demonstrate for the first time that TBI precipitates cognitive impairment in presymptomatic AD mice, while also confirming extracellular Aβ deposits following TBI. The recognition of this pathological link between TBI and AD should aid in developing novel treatments directed at abrogating cellular injury and extracellular Aβ deposition in the brain.

Downregulation of DAB2IP Promotes Mesenchymal-To-Neuroepithelial Transition and Neuronal Differentiation of Human Mesenchymal Stem Cells

The DOC-2/DAB2 interactive protein (DAB2IP) is a new member of the Ras GTPase–activating protein family. Recent studies have shown that, in addition to its tumor suppressive role in various tumors, DAB2IP also plays an important role in regulating neuronal migration and positioning during brain development. In this study, we determined the roles of DAB2IP in the neuronal differentiation of human mesenchymal stem cells (hMSCs). We found that lentiviral short hairpin RNA (shRNA)-mediated knockdown of DAB2IP promoted the mesenchymal-to-neuroepithelial stem cell transition (MtNeST) and neuronal differentiation, which were accompanied by a reduction of cell proliferation but not apoptosis or cellular senescence. This suggests that DAB2IP plays an important role in the neuronal induction of hMSCs. Moreover, our finding that reduction of glycogen synthase kinase 3 beta (GSK3β) activity upon LiCl pretreatment inhibited both the MtNeST and production of MAP2-positive cells upon DAB2IP knockdown suggests that this transition is most likely mediated by regulation of the GSK3β signaling pathway. Our study demonstrates that DAB2IP participates in the first step of neuron induction of hMSCs, which implies a potentially important role for DAB2IP in the MtNeST during neurogenesis.

Spatiotemporal changes in Cx30 and Cx43 expression during neuronal differentiation of P19 EC and NT2/D1 cells.

While connexins (Cxs) are thought to be involved in differentiation, their expression and role has yet to be fully elucidated. We investigated the temporal expression of Cx30, Cx36 and Cx43 in two in vitro models of neuronal differentiation: human NT2/D1 and murine P19 cells, and the spatial localisation of Cx30 and Cx43 in these models.A temporal Cx43 downregulation was confirmed in both cell lines during RA-induced neuronal differentiation using RT-PCR (P < 0.05) preceding an increase in neuronal doublecortin protein. RT-PCR showed Cx36 was upregulated twofold in NT2/D1 cells (P < 0.05) and sixfold in P19 cells (P < 0.001) during neuronal differentiation. Cx30 exhibited a transient peak in expression midway through the timecourse of differentiation increasing threefold in NT2/D1 cells (P < 0.001) and eightfold in P19 cells (P < 0.01).Qualitative immunocytochemistry was used to examine spatiotemporal patterns of Cx protein distribution alongside neuronal differentiation markers. The temporal immunolabelling pattern was similar to that seen using RT-PCR. Cx43 was observed intracellularly and on cell surfaces, while Cx30 was seen as puncta. Spatially Cx43 was seen on doublecortin-negative cells, which may indicate Cx43 downregulation is requisite for differentiation in these models. Conversely, Cx30 puncta were observed on doublecortin-positive and -negative cells in NT2/D1 cells and examination of the Cx30 peak showed puncta also localized to nestin-positive cells, with few puncta on MAP2-positive cells. In P19 cells Cx30 was localized on clusters of cells surrounded by MAP2- and doublecortin-positive processes. The expression pattern of Cx30 indicates a role in neuronal differentiation; the nature of that role warrants future investigation.

Unveiling the effects of the secretome of mesenchymal progenitors from the umbilical cord in different neuronal cell populations

It has been previously shown that the secretome of Human Umbilical Cord Perivascular Cells (HUCPVCs), known for their mesenchymal like stem cell character, is able to increase the metabolic viability and hippocampal neuronal cell densities. However, due to the different micro-environments of the distinct brain regions it is important to study if neurons isolated from different areas have similar, or opposite, reactions when in the presence of HUCPVCs secretome (in the form of conditioned media-CM). In this work we: 1) studied how cortical and cerebellar neuronal primary cultures behaved when incubated with HUCPVCs CM and 2) characterized the differences between CM collected at two different conditioning time points. Primary cultures of cerebellar and cortical neurons were incubated with HUCPVCs CM (obtained 24 and 96 h after three days of culturing). HUCPVCs CM had a higher impact on the metabolic viability and proliferation of cortical cultures, than the cerebellar ones. Regarding neuronal cell densities it was observed that with 24 h CM condition there were higher number MAP-2 positive cells, a marker for fully differentiated neurons; this was, once again, more evident in cortical cultures. In an attempt to characterize the differences between the two conditioning time points a proteomics approach was followed, based on 2D Gel analysis followed by the identification of selected spots by tandem mass spectrometry. Results revealed important differences in proteins that have been previously related with phenomena such as neurl cell viability, proliferation and differentiation, namely 14-3-3, UCHL1, hsp70 and peroxiredoxin-6. In summary, we demonstrated differences on how neurons isolated from different brain regions react to HUCPVCs secretome and we have identified different proteins (14-3-3 and hsp70) in HUCPVCs CM that may explain the above-referred results.

Oxygen-Controlled Automated Neural Differentiation of Mouse Embryonic Stem Cells

Automation and oxygen tension control are two tools that provide significant improvements to the reproducibility and efficiency of stem cell production processes. the aim of this study was to establish a novel automation platform capable of controlling oxygen tension during both the cell-culture and liquid-handling steps of neural differentiation processes. We built a bespoke automation platform, which enclosed a liquid-handling platform in a sterile, oxygen-controlled environment. An airtight connection was used to transfer cell culture plates to and from an automated oxygen-controlled incubator. Our results demonstrate that our system yielded comparable cell numbers, viabilities, metabolism profiles and differentiation efficiencies when compared with traditional manual processes. Interestingly, eliminating exposure to ambient conditions during the liquid-handling stage resulted in significant improvements in the yield of MAP2-positive neural cells, indicating that this level of control can improve differentiation processes. This article describes, for the first time, an automation platform capable of maintaining oxygen tension control during both the cell-culture and liquid-handling stages of a 2D embryonic stem cell differentiation process.

Patterning of human cord blood-derived stem cells on single cell posts and lines: Implications for neural commitment.

Using stem cells (SC) in new strategies for clinical treatment requires control of stem cell fate decision and the ability to govern their patterning and commitment in tissue engineering. Neural stem cells and other adult SC can respond to the different components of the microenvironment and their spatial arrangement in the stem cell niche. It has been shown previously by our group that different composition and architecture of patterned bioactive domains influence the developmental response of neural stem cells. In the present report we verify the commitment and differentiation of neural stem cells derived from human cord blood (HUCB-NSC) by a single cell patterning system. Microcontact printing technology was used to generate single cell positioning areas of different geometry: 10 micrometer-thin lines and 10 micrometer-width one cell posts. The commitment and differentiation of HUCB-NSC cells cultured on different surfaces were dependent on the geometry and the type of biomaterial present in bioactive domains. Fibronectin promoted neuronal protrusion outgrowth (Beta-tubulin III and MAP-2 positive cells) and gap junction development (Cx43 marker) between cells growing on lines while poly-L-lysine promoted HUCB-NSC differentiation into astrocytic, glial fibrillary acidic protein expressing (GFAP positive) cell phenotype. Here we also demonstrate by scanning electron microscopy that morphology of cells on the patterned surface is highly dependent upon the type of biomolecules used for printing. These kinds of platforms can be used for investigating the influence of spatial organization of environment on the SC fate decision and for studying the molecular processes occurring in a single cell.

Direct protection of neurons and astrocytes by matrine via inhibition of the NF-κB signaling pathway contributes to neuroprotection against focal cerebral ischemia

Matrine (Mat) and oxymatrine are two major alkaloids of the Chinese herb Sophora flavescens Ait. (Leguminosae). Previous study has demonstrated that Mat reduces brain edema induced by focal cerebral ischemia. More recently, oxymatrine has been reported to produce neuroprotective effects against focal cerebral ischemia via inhibiting the activation of NF-κB in the ischemic brain tissue. In the current study, we investigated whether direct protection on neurons and astrocytes via inhibition of NF-κB signaling pathway is associated with Mat's neuroprotective effects against cerebral ischemia. In a model of permanent middle cerebral artery occlusion (pMCAO), Mat (12.5, 25 and 50mg/kg) reduced the infarction volume and improved the neurological deficits in a dose-dependent manner, administered 10min, 3h and even 6h following pMCAO. Mat 50mg/kg also decreased the hemispheric water content. The number of GFAP-positive cells was markedly decreased in the ischemic cortex at 12h after ischemia. In contrast, Mat increased the number of GFAP-positive cells. Mat 50mg/kg has no effect on the cerebral blood flow (CBF). Primary neuron or astrocyte cultures were exposed to a paradigm of ischemic insult by using an oxygen–glucose deprivation (OGD), Mat (50–200μM) reduced LDH leakage and the number of neuronal and astrocytic apoptosis, and increased the number of MAP2-positive and GFAP-positive cells. Further observations revealed that Mat increased the protein levels of IκBα, and blocked the translocation of NF-κB p65 from the cytosol to the nucleus in the ischemic cortex and injured neurons and astrocytes induced by in vitro OGD. Moreover, Mat could down-regulate NF-κB p65 downstream pro-apoptotic gene p53 and/or c-Myc in the injured neurons and astrocytes induced by OGD. The present findings suggest that Mat, even when administrated 6h after ischemia, has neuroprotective effects against focal cerebral ischemia and directly protects neurons and astrocytes via inhibition of NF-κB signaling pathway, contributing to matrine's neuroprotection against focal cerebral ischemia.

Directing neuronal differentiation of primary neural progenitor cells by gene knockdown approach.

Directing differentiation of neural stem/progenitor cells (NPCs) to produce functional neurons is a promising remedy for neural pathological conditions. The major challenge, however, lies in the effective and efficient generation of a sizable population of neurons. A potential strategy is to incorporate RNA interference (RNAi) during directed stem cell differentiation to recapitulate the complex cell-signaling cascades that often occurs during the process. In this study, in vitro silencing of RE1-silencing transcription factor (REST) was carried out using small-interfering RNAs (siRNAs) to evaluate the efficacy of combining REST knockdown with conventional differentiation approaches to enhance neurogenesis. While earlier studies have demonstrated enhanced neuronal lineage commitment from embryonic stem cells and mesenchymal stem cells upon REST knockdown, the effects of REST silencing during other stages of neural development have not been extensively evaluated. We hypothesize that REST knockdown would enhance NPC development to mature neurons and that induced REST silencing can serve as a potential biochemical approach to direct cell fate. Under nonspecific induction conditions, REST knockdown induced eightfold higher Tuj1 mRNA expression at day 14 compared with untransfected cells and cells subjected to scrambled-siRNA treatment (controls). Immunostaining also revealed greater percentage of Tuj1 positive cells with REST knockdown. Combined with neuronal induction, REST silencing enhanced the kinetics of neuronal differentiation and the rate of maturation of committed neuronal cells. Specifically, upregulation of MAP2 occurred as early as 3 days after induction with REST silencing and the expression was comparable to the controls at day 14. Likewise, downregulation of REST generated more than twice the percentage of Tuj1 and MAP2 positive cells compared with controls at day 5 (p<0.05). Morphologically, REST-silencing enhanced the number and length of neurite extensions from Tuj1 positive cells (p<0.05), which was not evaluated in previous differentiation studies with REST knockdown. Taken together, these results demonstrate the efficacy of combining REST silencing during directed NPC differentiation to enhance the rate of differentiation and subsequent maturation of NPCs. This study also highlights the potential of RNAi as a biomedical strategy for guided stem cell differentiation.

Baicalin promotes neuronal differentiation of neural stem/progenitor cells through modulating p-stat3 and bHLH family protein expression

Signal transducer and activator of transcription 3 (stat3) and basic helix–loop–helix (bHLH) gene family are important cellular signal molecules for the regulation of cell fate decision and neuronal differentiation of neural stem/progenitor cells (NPCs). In the present study, we investigated the effects of baicalin, a flavonoid compound isolated from Scutellaria baicalensis G, on regulating phosphorylation of stat3 and expression of bHLH family proteins and promoting neuronal differentiation of NPCs. Embryonic NPCs from the cortex of E15–16 rats were treated with baicalin (2, 20μM) for 2h and 7days. Neuronal and glial differentiations were identified with mature neuronal marker microtubule associated protein (MAP-2) and glial marker Glial fibrillary acidic protein (GFAP) immunostaining fluorescent microscopy respectively. Phosphorylation of stat3 (p-stat3) and expressions of bHLH family genes including Mash1, Hes1 and NeuroD1 were detected with immunofluorescent microscopy and Western blot analysis. The results revealed that baicalin treatment increased the percentages of MAP-2 positive staining cells and decreased GFAP staining cells. Meanwhile, baicalin treatment down-regulated the expression of p-stat3 and Hes1, but up-regulated the expressions of NeuroD1 and Mash1. Those results indicate that baicalin can promote the neural differentiation but inhibit glial formation and its neurogenesis-promoting effects are associated with the modulations of stat3 and bHLH genes in neural stem/progenitor cells.

Intracranial spreading of a spinal anaplastic astrocytoma

A 63-year-old man was admitted with bilateral sensory loss up to thoracic level TH 8, progressive spastic paraparesis of legs, urinary incontinence, and fecal obstipation. Symptoms had begun 3 weeks earlier. Spinal MRI showed a contrast-enhancing mass with perifocal edema in the spinal cord at TH 6–7, and brain MRT was unsuspicious (Fig. 1a). Cerebral spinal fluid (CSF) revealed leukocytosis (19 cells/ll), elevated lactate (4.9 mmol/l), and protein (65.8 mg/dl) without neoplastic cells. Suspecting an autonomic inflammatory disease, steroids were applied, leading only to temporary improvement. Plasma separation brought no improvement. MRI then demonstrated an enlargement of the intramedullary mass with subtotal compression of the spinal cord. A biopsy showed astrocytic cells with GFAP (glial fibrillary acidic protein) and MAP2-positive tumor cells. The Ki67-proliferation index was beyond 5%. Diagnosis of an anaplastic astrocytoma WHO grade III was made. Since gross total resection was impossible, combined radiochemotherapy with temozolomide [1] was undertaken. Symptoms and MRI scans remained stable, but 14 months after the first symptoms, a progressive complete internal and external paresis of the left oculomotor nerve occurred. The other symptoms remained stable. Brain MRI revealed enlargement of the left oculomotor nerve in its prepontine course to the cavernous sinus with contrast enhancement as a sign of cortical infiltration of the adjacent temporomesial parenchyma (Fig. 1b). Spinal MRI showed cervical and thoracic leptomeningeal enhancement (Fig. 1c, d), the primary intramedullary tumor remained stable. CSF revealed elevated lactate (5.3 mmol/l) and lympho-monocytic pleocytosis (14 cells/ll) with atypic cells suspicious for meningeosis neoplastica. A biopsy of the swollen oculomotor nerve showed tumor cells with many mitoses, areas of necrosis, focal GFAP-positive cell accumulation (Online resource 1), and nuclear accumulation of p53 in a few cells. The Ki67 proliferation index was above 30%, therefore the histological evaluation was glioblastoma. Only a few days after the biopsy, the patient developed dysphagia, dysarthria, and dyspnea, which was most likely due to basal infiltration of the leptomeninges, and died within 2 weeks. In summary, the patient presented first with a spinal anaplastic astrocytoma WHO grade III. Later leptomeningeal and cerebral dissemination occurred. Histology revealed a growing malignancy consistent with glioblastoma. This case is of special interest because of the very rare circumstance of a malignant spinal anaplastic astrocytoma spreading to spinal and intracranial leptomeninges and even supratentorial parenchyma. The second biopsy revealed a different—higher—grade than before, therefore a multifocal glioma cannot be ruled out completely, but Electronic supplementary material The online version of this article (doi:10.1007/s00415-011-6253-0) contains supplementary material, which is available to authorized users.

Differentiation of human ES cell-derived neural progenitors to neuronal cells with regional specific identity by co-culturing of notochord and somite

Limitations to the in vivo study of human nervous system development make it necessary to design an in vitro model to evaluate the in vivo effects of surrounding tissues on neurogenesis and regional identity of the human neural plate. Rostral neural progenitors (NPs) were initially generated from adherent human embryonic stem cells (hESCs) in a defined condition and characterized. Then, to find the role of somites (S) and notochords (N) in rostro-caudal (RC) and dorso-ventral (DV) patterning of neuronal cells, NPs were co-cultured with microencapsulated chicken S or N in alginate beads. In this study, N induced more neurogenesis as evaluated by expression of TUJ1 and MAP2-positive cells. Additionally, N induced spinal cord ventral brachiothoracic identity as well as NPs proliferation. We observed a synergic effect on motoneuron induction when S and N were used together. Moreover, S induced hindbrain identity in differentiated neuronal cells from NPs. These results indicate that highly enriched NPs can be generated in an adherent and defined system from hESCs. Moreover, S and N tissues highly influenced neuronal differentiation in point of proliferation, neurogenesis, and RC and DV regional identity. These results indicate a very simple and efficient protocol to mimic in vivo events of human neural development in vitro which is important in the context of developmental neuroscience and cellular replacement therapies.

CAMP promotes differentiation of rodent neuronal progenitor cells

Numerous studies have described neuronal differentiation of neural progenitor cells derived from fetal tissue in vitro, but the biochemical mechanisms underlying this process remain largely unknown. In the present study, the role of cAMP in promoting functional maturation of neuronal progenitor cells (NPCs) from the subventricular zone (SVZ) of rodent fetal brain was investigated. NPCs were extracted from telencephalic vesicles of E14 rat embryos and then expanded in medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). A mature neuronal fate was induced by: i) withdrawal of growth factors (basal condition); ii) addition of brain-derived neurotrophic factor (BDNF); or iii) addition of isobutylmethylxantine (IBMX). Whole cell patch clamping assessed electrophysiological properties. Immunocyto - chemistry for MAP2 confirmed neuronal differentiation. Quantification of neuronal cells, and determination of their electrophysiological properties, was performed in the cited experimental groups. IBMX significantly enhanced the yield of MAP2-positive neurons in the culture system (3.7-fold increase). Application of a one-week differentiation protocol under IBMX induced functional maturity. MAP2-positive cells presented large Na- and K- voltagedependent currents, fired bursts of action potentials, and exhibited spontaneous synaptic activity. Further, IBMX proved more effective than BDNF for promoting neuronal maturation, leading to higher evoked peak currents and current densities, as well as greater yields of cells firing action potentials and presenting active synaptic contacts. Our data indicates the importance of cAMP-dependent mechanisms for maturation of neuronal progenitor cells in vitro. This knowledge can enable future manipulation of neurogenesis in vivo in order to promote the intrinsic regenerative capacity of the central nervous system.