Neurobasal Medium Research Articles

Promoting motor functions in a spinal cord injury model of rats using transplantation of differentiated human olfactory stem cells: A step towards future therapy

Human olfactory ecto-mesenchymal stem cells (hOE-MSCs) derived from the human olfactory mucosa (OM) can be easily isolated and expanded in cultures while their immense plasticity is maintained. To mitigate ethical concerns, the hOE-MSCs can be also transplanted across allogeneic barriers, making them desirable cells for clinical applications. The main purpose of this study was to evaluate the effects of administering the hOE-MSCs on a spinal cord injury (SCI) model of rats. These cells were accordingly isolated and cultured, and then treated in the neurobasal medium containing serum-free Dulbecco’s Modified Essential Medium (DMEM) and Ham’s F-12 Medium (DMEM/F12) with 2% B27 for two days. Afterwards, the pre-induced cells were incubated in N2B27 with basic fibroblast growth factor (bFGF), fibroblast growth factor 8b (FGF8b), sonic hedgehog (SHH), and ascorbic acid (vitamin C) for six days. The efficacy of the induced cells was additionally evaluated using immunocytochemistry (ICC) and real-time polymerase chain reaction (RT-PCR). The differentiated cells were similarly transplanted into the SC contusions. Functional recovery was further conducted on a weekly basis for eight consecutive weeks. Moreover, cell integration was assessed via conventional histology and ICC, whose results revealed the expression of choline acetyltransferase (ChAT) marker at the induction stage. According to the RT-PCR findings, the highest expression level of insulin gene-enhancer protein (islet-1), oligodendrocyte transcription factor (Olig2), and homeobox protein HB9 was observed at the induction stage. The number of engraftment cells also rose (approximately by 2.5 % ± 0.1) in the motor neuron-like cells derived from the hOE-MSCs-grafted group compared with the OE-MSCs-grafted one. The functional analysis correspondingly revealed that locomotor and sensory scores considerably improved in the rats in the treatment group. These findings suggested that motor neuron-like cells derived from the hOE-MSCs could be utilized as an alternative cell-based therapeutic strategy for SCI.

Aptamer Conformational Change Enables Serotonin Biosensing with Nanopipettes.

We report artificial nanopores in the form of quartz nanopipettes with ca. 10 nm orifices functionalized with molecular recognition elements termed aptamers that reversibly recognize serotonin with high specificity and selectivity. Nanoscale confinement of ion fluxes, analyte-specific aptamer conformational changes, and related surface charge variations enable serotonin sensing. We demonstrate detection of physiologically relevant serotonin amounts in complex environments such as neurobasal media, in which neurons are cultured in vitro. In addition to sensing in physiologically relevant matrices with high sensitivity (picomolar detection limits), we interrogate the detection mechanism via complementary techniques such as quartz crystal microbalance with dissipation monitoring and electrochemical impedance spectroscopy. Moreover, we provide a novel theoretical model for structure-switching aptamer-modified nanopipette systems that supports experimental findings. Validation of specific and selective small-molecule detection, in parallel with mechanistic investigations, demonstrates the potential of conformationally changing aptamer-modified nanopipettes as rapid, label-free, and translatable nanotools for diverse biological systems.

195 Development of a fetal porcine model for congenital viral infections in the developing CNS

To develop a porcine model for analysis of the effects of congenital viral infections on CNS and spinal (CSF) derived fetal neural precursor (NPC) and neural progenitor cells. Isolation of NPCs from the subventricular zone (SVZ) of the porcine brain or CSF derived from premature (day 104) and post-term (day 135) Cesarean-delivered piglets were plated in 12-well plates. Nonadherent plates were used to generate neurospheres with self-renewal capacity and for long term passages. After a 2-day incubation for neurosphere formation and removal of apoptotic debris, cells were seeded in various conditions including incubation in (1) 10% FBS, (2) Rock Inhibitor (RI)-specific inhibitor of apoptosis, (3) FBS and RI, and (4) Neurobasal media alone (control). After purification and identification of NPCs, cells were then infected with low MOI of clinically isolated first passage ZIKV HN16, then fixed and stained with Flavivirus-specific antibody (MAb 4G2) followed by FITC labeled secondary antibody staining visualization under fluorescence microscopy. Gene expression studies were also performed from RNA from ZIKV infected and mock infected samples to determine effects on genes related to NPC self-renewal and survival. The brain derived cells cultured for 4 days showed three distinct populations including NSCs, NPCs, and a mixed NSC/NPC population. After 21 days of culture, cells shifted from an NSC population toward later within the NPC lineage. Both brain and CSF cells showed relatively equal permissivity to ZIKV viral infection (Fig.1). Moreover, there was no difference in Vimentin gene expression in ZIKV infected cells, while SOX-2 and Nestin gene expression following ZIKV infection was significantly lower (p<0.05; Fig.2). The findings that Nestin expression is downregulated when comparing brain and CSF cells, particularly after ZIKA infection, is of interest since Nestin is postulated to play a role in axonal growth. This study demonstrated the ability to utilize porcine fetal NPCs in a safe and effective manner for the study of NPC development and effects of viral infections

Comparison of Rat Primary Midbrain Neurons Cultured in DMEM/F12 and Neurobasal Mediums.

Introduction:Midbrain dopaminergic neurons are involved in various brain functions, including motor behavior, reinforcement, motivation, learning, and cognition. Primary dopaminergic neurons and also several lines of these cells are extensively used in cell culture studies. Primary dopaminergic neurons prepared from rodents have been cultured in both DMEM/F12 and neurobasal mediums in several studies. However, there is no document reporting the comparison of these two mediums. So in this study, we evaluated the neurons and astroglial cells in primary midbrain neurons from rat embryos cultured in DMEM/F12 and neurobasal mediums.Methods:Primary mesencephalon cells were prepared from the E14.5 rat embryo. Then they were seeded in two different mediums (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 [DMEM/F12] and neurobasal). On day 3 and day 5, half of the medium was replaced with a fresh medium. On day 7, β3-tubulin-, GFAP (Glial fibrillary acidic protein)- and Tyrosine Hydroxylase TH-positive cells were characterized as neurons, astrocytes, and dopaminergic neurons, respectively, using immunohistochemistry. Furthermore, the morphology of the cells in both mediums was observed under light microscopy on days 1, 3, and 5.Results:The cells cultured in both mediums were similar under light microscopy regarding the cell number, but in a neurobasal medium, the cells have aggregated and formed clustering structures. Although GFAP-immunoreactive cells were lower in neurobasal compared to DMEM/F12, the number of β3-tubulin- and TH-positive cells in both cultures was the same.Conclusion:This study’s findings demonstrated that primary midbrain cells from the E14.5 rat embryo could grow in both DMEM/F12 and neurobasal mediums. Therefore, considering the high price of a neurobasal medium, it can be replaced with DMEM/F12 for culturing primary dopaminergic neurons.

Effect of Neural Stem Cells Transplantation on the Paradoxical Sleep in the Rat

Introduction: Nowadays a lot of research is being done on neural stem cell (NSCs) transplantation to repair brain damage. Neural stem cells are found in the nervous system. These cells can differentiate interneurons, astrocytes, and oligodendrocytes. Noradrenergic cells in the locus coeruleus (LC) play an important role in paradoxical sleep (PS). The purpose of this study was the assessment of NSCs transplantation on paradoxical sleep in the unilateral lesion of LC. Methods: Forty adult male Wistar rats (225-250 gr), were categorized into four groups (Control, Lesion, experimental 1 (intravenous transplantation of NSCs), and experimental 2 (intravenous transplantation of NACs). NSCs were obtained from the subventricular zone (SVZ) of newborn rat brains. NSCs were differentiated into NACs in neurobasal medium, B-27, GDNF (30 ng/ ml) and BDNF (50 ng/ ml) for 5 days. We used 3,000,000 cells for cell transplantation. The animals received unilateral lesion of LC by injection of 6_hydroxydopamine. For paradoxical sleep recording, 2EMG and 3 EEG electrodes were placed in the neck and skull muscles, respectively. Results: In this study Tyrosine Hydroxylase (TH) was detected in NACs. A significant increase in paradoxical sleep was seen in the lesion group in comparison with the control group. After NSCs and NACs transplantation, a significant decrease in PS was seen in experimental groups in comparison with the lesion group. Conclusion: The results show that NSCs and NACs transplantation improve PS after unilateral lesion of LC.

Neural crest stem cells can be induced in vitro from human-induced pluripotent stem cells using a novel protocol free of feeder cells.

Objective: Our knowledge of human neural crest stem cells (NCSCs) is expanding, owing to recent advances in technologies utilizing human-induced pluripotent stem cells (hiPSCs) that generate NCSCs. However, the clinical application of these technologies requires the reduction of xeno-materials. To overcome this significant impediment, this study aimed to devise a novel method to induce NCSCs from hiPSCs without using a feeder cell layer.Materials and Methods: hiPSCs were cultured in feeder-free maintenance media containing the Rho-associated coiled-coil forming kinase inhibitor Y-27632. When the cells reached 50–70% confluence, differentiation was initiated by replacing the medium with knockout serum replacement (KSR) medium containing Noggin and SB431542. The KSR medium was then gradually replaced with increasing concentrations of Neurobasal medium from day 5 to 11.Results: Immunocytochemistry and flow cytometry were performed 12 days after induction of differentiation and revealed that the cells generated from hiPSCs expressed the NCSC markers p75 and HNK-1, but not the hiPSC marker SOX2.Conclusion: These findings demonstrate that hiPSCs were induced to differentiate into NCSCs in the absence of feeder cells.

Co-expression of cancer driver genes: IDH-wildtype glioblastoma-derived tumorspheres

BackgroundDriver genes of GBM may be crucial for the onset of isocitrate dehydrogenase (IDH)-wildtype (WT) glioblastoma (GBM). However, it is still unknown whether the genes are expressed in the identical cluster of cells. Here, we have examined the gene expression patterns of GBM tissues and patient-derived tumorspheres (TSs) and aimed to find a progression-related gene.MethodsWe retrospectively collected primary IDH-WT GBM tissue samples (n = 58) and tumor-free cortical tissue samples (control, n = 20). TSs are isolated from the IDH-WT GBM tissue with B27 neurobasal medium. Associations among the driver genes were explored in the bulk tissue, bulk cell, and a single cell RNAsequencing techniques (scRNAseq) considering the alteration status of TP53, PTEN, EGFR, and TERT promoter as well as MGMT promoter methylation. Transcriptomic perturbation by temozolomide (TMZ) was examined in the two TSs.ResultsWe comprehensively compared the gene expression of the known driver genes as well as MGMT, PTPRZ1, or IDH1. Bulk RNAseq databases of the primary GBM tissue revealed a significant association between TERT and TP53 (p < 0.001, R = 0.28) and its association increased in the recurrent tumor (p < 0.001, R = 0.86). TSs reflected the tissue-level patterns of association between the two genes (p < 0.01, R = 0.59, n = 20). A scRNAseq data of a TS revealed the TERT and TP53 expressing cells are in a same single cell cluster. The driver-enriched cluster dominantly expressed the glioma-associated long noncoding RNAs. Most of the driver-associated genes were downregulated after TMZ except IGFBP5.ConclusionsGBM tissue level expression patterns of EGFR, TERT, PTEN, IDH1, PTPRZ1, and MGMT are observed in the GBM TSs. The driver gene-associated cluster of the GBM single cells were enriched with the glioma-associated long noncoding RNAs.

Preparation of Adhesion Culture of Neural Stem/Progenitor Cells of the Olfactory Mucosa for the Treatment of Spinal Cord Injuries.

In this work, an optimal protocol was developed for obtaining adhesion culture of neural stem/progenitor cells (NSPC) of rat olfactory mucosa. During the development of the protocol, the conditions for cell culturing on adhesion substrates fibronectin and laminin in DMEM/F-12 and neurobasal media with the same culture additives were compared. Cell proliferation was maximum during culturing on both substrates in the neurobasal medium. Using the immunofluorescence method, we found that culturing on fibronectin in the neurobasal medium ensured maximum (52.22%) content of nestin-positive cells in comparison with other culturing conditions. The highest percentage of βIII-tubulin-positive cells was detected in cultures growing on fibronectin in the neurobasal medium and in DMEM/F-12 (79.11 and 83.52%, respectively). Culturing in adhesion cultures in the neurobasal medium on fibronectin allowed obtaining cultures enriched with NSPC and neurons differentiating from them in a quantity sufficient for further transplantation. The developed protocol can be recommended for obtaining NPSC from human olfactory mucosa for the treatment of spinal cord injuries.

The Neurotrophic Function of Glucagon-Like Peptide-1 Promotes Human Neuroblastoma Differentiation via the PI3K-AKT Axis.

Simple SummaryThe study demonstrated that the treatment with GLP-1 of SH-SY5Y human neuroblastoma cells increased the expression of AMPA receptors, NMDA receptors, dopamine receptors, synaptic proteins-synapsin 1, synaptophysin, and postsynaptic density protein 95, but not muscular and nicotinic acetylcholine receptors. In addition, the biomarker of dividing neuronal cells, vimentin, was decreased after treatment with GLP-1. Tuj1 immunostaining images showed that GLP-1 induced neurite processes and the development of neuronal morphologies. The GLP-1-differentiated neurons were able to be induced to generate action potentials by single cell patch-clamp. Our results also suggested that the PI3K-AKT axis is the dominant signaling pathway promoting the differentiation of SH-SY5Y cells into mature and functional neurons in response to GLP-1 receptor activation. The sequential treatment of retinoic acid and GLP-1 within a serum-free medium is able to trigger the differentiation of SH-SY5Y cells into morphologically and physiologically mature glutamatergic and dopaminergic neurons.Background: Neurons are terminally-differentiated cells that generally develop from neuronal stem cells stimulated by various neurotrophic factors such as NGF, BDNF, NT3, and NT-4. Neurotrophic factors have multiple functions for neurons, including enabling neuronal development, growth, and protection. Glucagon-like peptide-1 (GLP-1) is an intestinal-secreted incretin that enhances cellular glucose up-take to decrease blood sugar levels. However, many studies suggest that the function of GLP-1 is not limited to the regulation of blood sugar levels. Instead, it may also act as a neurotrophic factor with a role in ensuring neuronal survival and neurite outgrowth, as well as protecting synaptic plasticity and memory formation. Methods: The SH-SY5Y cells were differentiated by sequential treatments of retinoic acid and GLP-1 treatment within polyethylenimine-coated dishes under serum-free Neurobasal medium. PI3K inhibitor (LY294002) and MEK inhibitor (U0126) were used to determine the signaling pathway in regulation of neuronal differentiation. Neuronal marker (TUJ1) and synaptic markers (synapsin 1, synaptophysin, and PSD95) as well as single cell patch-clamp were applied to determine maturity of neurons. Antibodies of AMPA receptor, NMDA receptor subunit 2A, dopamine receptor D1, muscarinic acetylcholine receptor 2, and nicotinic acetylcholine receptor α4 were used to examine the types of differentiated neurons. Results: Our study’s results demonstrated that the treatment with GLP-1 of SH-SY5Y human neuroblastoma cells increased the expression of AMPA receptors, NMDA receptors, dopamine receptors, synaptic proteins-synapsin 1, synaptophysin, and postsynaptic density protein 95, but not muscular and nicotinic acetylcholine receptors. In addition, the biomarker of dividing neuronal cells, vimentin, was decreased after treatment with GLP-1. Tuj1 immunostaining images showed that GLP-1 induced neurite processes and the development of neuronal morphologies. The GLP-1-differentiated neurons were able to be induced to generate action potentials by single cell patch-clamp. Our study also suggested that the PI3K-AKT axis is the dominant signaling pathway promoting the differentiation of SH-SY5Y cells into mature and functional neurons in response to GLP-1 receptor activation. Conclusions: The sequential treatment of retinoic acid and GLP-1 within a serum-free medium is able to trigger the differentiation of SH-SY5Y cells into morphologically and physiologically mature glutamatergic and dopaminergic neurons.

Neuronal transcription program induced in hippocampal cells cocultured with bone marrow derived mesenchymal cells

Several approaches have been applied to harvest bone marrow stromal cells (BMSCs) and to differentiate into neurons or neuronal-like cells through chemical stimulation or exposing to growth factors. To date, the data regarding induction or regulation of neuronal transcription program in neuronal-like cells derived from BMSCs is yet unknown. The objective of this study is to co-culture BMSCs with neonatal hippocampal cells and generate neuronal-like cells by direct cell-to-cell contact system without using neuronal growth factors or neurobasal medium. Here, we proposed a role for NeuroD1 and Neurogenin -2 bHLH family of transcription factors implicated in onset of neurogenesis and differentiation of cells into neurons in promoting the interaction of hippocampal cells with BMSCs and their differentiation in to neurons. The proliferation of the cells was assessed with MTT assay and the role of neuronal induction and differentiation transcription regulators NeuroD1 and Neurogenin-2 in cocultured cells was determined through immunocytochemical analysis. We observed activation and expression of the neurogenic transcription factors NeuroD1 and NGN-2 associated with neuronal activation program to initiate the onset of neurogenesis in cocultured cells. Further, our results have shown a significant expression of neuronal progenitor and immature neuronal marker i.e., nestin and tubulin respectively in cocultured cells endorsing the initiation of neuronal activation.

Age of the donor affects the nature of in vitro cultured human dental pulp stem cells

ObjectivesThe dental pulp stem cells (DPSCs) of six donors (three young donors aged < 19 years and three adult donors aged > 25 and < 30 years) were characterized for their stem cell marker expression and differentiation potential to study the effect of donor age on DPSCs in vitro. MethodsDPSCs were cultured in αMEM supplemented with 20% fetal calf serum (conventional conditions) or on fibronectin-coated flasks with neurobasal medium supplemented with B27, bFGF and EGF (alternative conditions). DPSCs were characterized by immunofluorescence staining to detect the neural crest/mesenchymal stem cells markers P75 and CD146, respectively. The differentiation potential was tested by the induction of DPSCs into osteogenic, adipogenic and glial lineages and then by detecting the corresponding markers osteocalcin, lipidtox and S100ß, respectively. ResultsThe DPSCs of the young donors expressed CD146 only under the conventional conditions and expressed P75 regardless of the culture conditions. However, the DPSCs of adult donors expressed CD146 only under the alternative conditions and expressed P75 only under conventional conditions. Only the DPSCs of the young donors differentiated into the glial linage. The DPSCs of the adult donors differentiated more efficiently into the adipogenic linage. Osteogenic differentiation was comparable. ConclusionDonor age affects the expression of stem cell markers and differentiation potential of DPSCs. Moreover, the effect of culture conditions on DPSCs is age dependent.

Roles and Interaction of the MAPK Signaling Cascade in Aβ25-35-Induced Neurotoxicity Using an Isolated Primary Hippocampal Cell Culture System.

Alzheimer's disease (AD) is characterized with increased formation of amyloid-β (Aβ) in the brain. Aβ peptide toxicity is associated with disturbances of several intracellular signaling pathways such as mitogen activated protein kinases (MAPKs). The aim of this study was to investigate the role of MAPKs and their interactions in Aβ-induced neurotoxicity using isolated hippocampal neurons from the rat. Primary hippocampal cells were cultured in neurobasal medium for 4days. Cells were treated with Aβ25-35 and/or MAPKs inhibitors for 24h. Cell viability was determined by an MTT assay and phosphorylated levels of P38, JNK, and ERK were measured by Western blots. Aβ treatment (10-40µM) significantly decreased hippocampal cell viability in a dose-dependent manner. Inhibition of P38 and ERK did not restore cell viability, while JNK inhibition potentiated the Aβ-induced neurotoxicity. Compared to the controls, Aβ treatment increased levels of phosphorylated JNK, ERK, and c-Jun, while it had no effect on levels of phosphorylated P38. In addition, P38 inhibition led to decreased expression levels of phosphorylated ERK; inhibition of JNK resulted in decreased expression of c-Jun; and inhibition of ERK, decreased phosphorylated levels of JNK. These results strongly suggest that P38, ERK, and JNK are not independently involved in Aβ-induced toxicity in the hippocampal cells. In AD, which is a multifactorial disease, inhibiting a single member of the MAPK signaling pathway, does not seem to be sufficient to mitigate Aβ-induced toxicity and thus their interactions with each other or potentially with different signaling pathways should be taken into account.

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Roles of Vitamin E in Energy Metabolism During Neurite Outgrowth

Abstract Objectives Neurite outgrowth is a pivotal process of brain development and recovery after brain injury. This metabolically demanding process requires assembly of the cytoskeleton and formation and maintenance of synapses. We have recently found that treatment with alpha-tocotrienol, an antioxidant and a member of the vitamin E family, prevents loss of mitochondrial inner membrane potential during oxidative stress. In this study, we hypothesize that the mitochondrion is the central target of alpha-tocotrienol-mediated neuroprotection, and treatment with alpha-tocotrienol may improve neuronal energy metabolism and promote neurite outgrowth. Methods Primary hippocampal neurons were grown in neurobasal media with or without alpha-tocotrienol for 3 weeks. Then, the morphological development of neurites, including polarity and arborization, was analyzed. We also assayed the ATP: ADP ratio in neurites using PercevalHR fluorescence biosensor after treatment with alpha-tocotrienol. Results Neurons grown with alpha-tocotrienol achieved neuronal polarity prior to the control group, and alpha-tocotrienol treated neurons showed longer and more branched neurites compared to the control group. Treatment with alpha-tocotrienol enhanced ATP levels in primary hippocampal neurons. Conclusions Our data show that alpha-tocotrienol improves mitochondria-mediated ATP production and supports the metabolically demanding process of neurite growth. This study also suggests that alpha-tocotrienol may be beneficial for recovery after brain injuries associated with neurite loss. Funding Sources RG14811 (University of Alabama).

Membrane Elastic Properties During Neural Precursor Cell Differentiation.

Neural precursor cells differentiate into several cell types that display distinct functions. However, little is known about how cell surface mechanics vary during the differentiation process. Here, by precisely measuring membrane tension and bending modulus, we map their variations and correlate them with changes in neural precursor cell morphology along their distinct differentiation fates. Both cells maintained in culture as neural precursors as well as those plated in neurobasal medium reveal a decrease in membrane tension over the first hours of culture followed by stabilization, with no change in bending modulus. During astrocyte differentiation, membrane tension initially decreases and then increases after 72 h, accompanied by consolidation of glial fibrillary acidic protein expression and striking actin reorganization, while bending modulus increases following observed alterations. For oligodendrocytes, the changes in membrane tension are less abrupt over the first hours, but their values subsequently decrease, correlating with a shift from oligodendrocyte marker O4 to myelin basic protein expressions and a remarkable actin reorganization, while bending modulus remains constant. Oligodendrocytes at later differentiation stages show membrane vesicles with similar membrane tension but higher bending modulus as compared to the cell surface. Altogether, our results display an entire spectrum of how membrane elastic properties are varying, thus contributing to a better understanding of neural differentiation from a mechanobiological perspective.

Hormetic effects of mild heat stress on the primary culture of mouse prefrontal cerebrocortical neurons

BackgroundHormesis is an adaptive response of a biological system to low dose of stressors. It exerts beneficial effects to enable the cells and organisms to sustain the unfavourable conditions. Mild heat stress is one of the widely studied hormetic agents having antiageing and lifespan prolonging effects. In order to study the effect of mild heat stress on the primary culture of mouse prefrontal cerebrocortical neurons with reference to ageing-associated degenerative alterations the present investigations were carried out.Study designThe prefrontal cerebrocortical neurons of E17 day mouse embryo were cultured on poly-l-lysine coated coverslips and fed with neurobasal medium supplemented with B-27 at 37 °C and 5% CO2. The neurons were divided into two groups: control group and mild heat stress group. The neurons from the control group were incubated at 37 °C without any heat stress. The neurons from the mild heat stress group were subjected to hyperthermic stress of 38 °C for 30 min on 2nd, 4th and 6th day of seeding.MethodsOn the 3rd, 5th and 7th day of incubation, viability was studied by calcein-AM and propidium iodide assay and cell death assay was carried out by lactate dehydrogenase (LDH) assay. The surviving neurons were enumerated on 10th, 15th and 20th day of incubation by live cell imaging. All microscopic studies were carried out by observer blinding.ResultsIt was observed that there was higher percentage of viable neurons and lower percentage of degenerating neurons in the mild heat stress group than the control. The difference was highly significant (p < 0.001).ConclusionMild heat stress (38 °C for 30 min on every alternate day up to 6 days of incubation) exerts hormetic effects on the primary culture of mouse prefrontal cerebrocortical neurons by delaying the degenerative alterations.

Prorenin Induces Intracellular Signaling And Reactive Oxygen Species In The Brainstem

The importance of the brain renin angiotensin system (RAS) in blood pressure (BP) regulation and water homeostasis is well accepted. However, it remains unclear whether renin and/or its precursor, prorenin, are expressed in the brain and whether they trigger signaling pathways in specific brain regions. We previously used double transgenic mice expressing enhanced green fluorescent protein controlled by renin promoter and beta‐galactosidase controlled by the human angiotensinogen (AGT) promoter to localize their expression in the brain. We reported that neurons expressing renin are localized in close proximity to AGT‐expressing cells in the rostral ventrolateral medulla (RVLM), a brainstem nucleus involved in sympathetic activity and BP control. Here, we hypothesized that prorenin induces phosphorylation of intracellular cascades such as extracellular signal‐regulated kinases (ERK1/2) and activation of nicotinamide adenine dinucleotide phosphate oxidase (NADPHox) in the brainstem. Neonatal brainstem cells were cultured from C57BL/6J mice at postnatal day 1–3 and maintained for 7–10 days in B27‐supplemented neurobasal medium. Immunofluorescence experiments demonstrated the cultures contain both MAP2+ neurons and GFAP+ astrocytes. Expression of type 1 angiotensin receptor (AT1R), renin, and prorenin receptor (PRR) was confirmed by qPCR. To quantify ERK1/2 phosphorylation, cells were stimulated with recombinant prorenin (rProrenin) at 100 nM for 0, 30, and 60 min. Total and phosphorylated ERK1/2 were detected by Western Blot. The effect of rProrenin on NADPHox activity was measured by lucigenin chemiluminescence assay. Specific activity of NADPHox was expressed as chemiluminescence units per min per microgram of protein. Vehicle and phorbol myristate acetate were used as negative and positive controls, respectively. Incubation with rProrenin increased the ratio of phosphorylated‐to‐total ERK1/2 from 0.65±0.07 A.U. at baseline to 1.01±0.13 A.U. at 30 min (54% increase; p=0.0035; n=7) and 1.29±0.09 A.U. at 60 min (102% increase; p&lt;0.0001; n=7). We generated preliminary data showing that both PRO20, a PRR blocker, and Ro‐31, a protein kinase C inhibitor, abrogated prorenin‐induced ERK1/2 phosphorylation. In an additional experiment, we observed that Angiotensin II (Ang II) at 100 nM induced ERK1/2 phosphorylation from 0.98 A.U. to 1.52 A.U. at 15 min (55 % increase), 1.61 A.U. at 30 min (65% increase), and 1.35 A.U. at 60 min (38% increase). Incubation of cells with 100 nM rProrenin for 6 hours resulted in 47.2±9.5 % induction of NADPHox activity compared to vehicle control (p&lt;0.05, n=5). Finally, fluorescent in situ hybridization (RNAscope) data suggests that PRR is expressed in the RVLM neurons and RVLM‐targeted stereotactic microinjection of glutamate, rProrenin, or Ang II, but not vehicle, induced an acute BP elevation in isoflurane‐anesthetized mice. We conclude that prorenin in the brainstem induces downstream signaling involving ERK/2 phosphorylation and generation of ROS, which might contribute to BP elevation and sympathoexcitation. Future studies will determine whether prorenin‐elicited signaling in the brainstem requires binding to PRR, generation of Ang II, and/or AT1R.Support or Funding InformationP01 HL084207 to Sigmund and 5T32HL134643 to Nakagawa

In Vitro Differentiation of Chicken Astrocytes: Growth, Morphology, and Protein Expression of Astrocytes in Primary Cultures.

Astrocytes regulate synaptic transmission in the central nervous system. Astrocytes in vivo have "stems" that express glial fibrillary acidic protein (GFAP), intermediate filaments, and peripheral astrocyte processes (PAPs), which contain actin-rich cytoskeletal structures. At the PAPs, the perisynaptic glia contacts and enwraps synapses, and modulates glia-neuronal communication. Cultured astrocytes have been an invaluable tool for studying roles of astrocytes; however, the morphology of mammalian primary astrocytes cultured in conventional medium containing fetal bovine serum (FBS) was similar to that of fibroblasts, and many culture conditions have been developed to generate stellate astrocytes observed in vivo. Avian astrocytes have been prepared from embryonic chick forebrain and maintained at a high cell density in conventional FBS-containing medium as mammalian astrocytes, thus the morphological analysis of chicken astrocytes has not yet been performed. In the present study, we report that the morphology of astrocytes freshly harvested from the forebrain of a chicken embryo in serum-free Neurobasal medium with B-27 supplement and basic fibroblast growth factor (bFGF) is similar to that of the astrocyte morphology in vivo. We also find that astrocytes in this medium express similar levels of GFAP and two actin-binding proteins as astrocytes in conventional FBS-containing medium, although they have different morphologies. Furthermore, we confirmed that cryopreserved astrocytes differentiate faster than freshly harvested astrocytes.

P3‐061: ALLOPREGNANOLONE POTENTIATES BIOENERGETIC CAPACITY AND RESTRUCTURES MITOCHONDRIAL RETICULUM IN NEURONS AND ASTROCYTES

We reported previously that the neurosteroid allopregnanolone (Allo) promotes neural stem cell regeneration, reverses neurogenic, metabolic and cognitive deficits and reduces Alzheimer's disease (AD) pathology in a mouse model of AD. To compare and contrast the cell-type specific mechanisms of Allo in regulating brain energy metabolism, we assessed the effect of Allo on mitochondrial bioenergetic profile and their morphological changes in rat hippocampal neurons and astrocytes. E18 rat hippocampal neurons were cultured for 10 days in neurobasal medium (NBM) with 2% B27 and then starved in 0.2% B27 / NBM for 4 hours before treatment with either 100nM Allo or 0.001% Vehicle overnight. E18 rat hippocampal astrocyte were cultured for 10 days in DMEM:F12(1:1) with 10% FBS and then starved in 10% Charcoal stripped-FBS / DMEM:F12 for 24 hours before treatment with 100nM Allo or 0.001% Vehicle overnight. Upon completion of treatment, both cell types were subject to morphological, biochemical and metabolic characterization of their mitochondrial phenotypes. In primary hippocampal neurons, Allo treatment significantly reversed nutrient deprivation-induced deficits in mitochondrial maximal- and spare respiratory capacity and dendritic morphology. In parallel, in primary hippocampal astrocytes, Allo rescued serum depletion-induced decline in mitochondrial spare respiratory capacity. Allo treatment reduced the population of less efficient swollen globule mitochondria in both neurons and astrocytes. Specifically in astrocytes, Allo decreased the number of hyperfused tubule mitochondria and increased small globule mitochondria. The effect of Allo on re-structuring nutrient or serum depletion-induced mitochondrial reticulum in both neurons and astrocytes was further supported by the restoration of the balance between Drp1 and Opa1 expression, which are key regulators for mitochondrial fission and fusion, respectively. Outcomes of our findings further support the promising therapeutic effects of Allo against bioenergetic deficits and mitochondrial inefficiency that emerge in early phases of AD, with mitochondrial dynamics being a potentially key targeted mechanism.

Directed differentiation of human adipose tissue-derived stem cells to dopaminergic neurons in low-serum and serum-free conditions

Directing the fate of mesenchymal stem cells (MSCs) to dopaminergic neurons has great importance in both biomedical studies and cell therapy of Parkinson’s disease. We recently generated dopamine-secreting cells from human adipose tissue-derived stem cells (hADSCs) by exposing the cells to a growth factor cocktail composed of SHH, bFGF, FGF8 and BDNF in low-serum condition. In the current study, we induced the cells by the same dopaminergic inducing cocktail in serum-free B27-supplemented Neurobasal medium. ADSCs differentiated in both conditions expressed several neuronal and dopaminergic markers. However, there were higher gene expression levels under the serum-free condition. Higher levels of TUJ1 and TH proteins were also detected in the cells exposed to the dopaminergic-inducing cocktail under serum-free Neurobasal condition. TH protein was expressed in about 28% and 60% of the cells differentiated in the low-serum and serum-free Neurobasal media, respectively. Moreover, the cells exposed to the dopaminergic-inducing cocktail in the serum-free Neurobasal condition released a more significant amount of dopamine in response to KCl-induced depolarization. Altogether, these findings show a greater efficiency of the serum-free Neurobasal condition for growth factor-directed differentiation of hADSCs to functional dopamine-secreting cells which may be valuable for transplantation therapy of Parkinson’s disease in future.