Rat Cortical Neuronal Cells Research Articles

RCOR1 improves neurobehaviors and neuron injury in rat cerebral palsy by Endothelin-1 targeting-induced Akt/GSK-3β pathway upregulation

BackgroundRE1 Silencing Transcription factor (REST) corepressor 1 (RCOR1) has been reported to orchestrate neurogenesis, while its role in cerebral palsy (CP) remains elusive. Besides, RCOR1 can interact with Endothelin-1 (EDN1), and EDN1 expression is related to brain damage. Therefore, this study aimed to explore the effects of RCOR1/EDN1 on brain damage during the progression of CP. MethodsCP rats were established via hypoxia–ischemia insult, and injected with lentivirus-RCOR1, followed by examination of brain pathological conditions. The RCOR1 and EDN1 interaction was recognized using hTFtarget. Healthy rat cortical neuron cells received interference of RCOR1/EDN1 expression, and underwent oxygen-glucose deprivation/reoxygenation (OGD/R) treatment, after which phenotypic and molecular assays were conducted through the biochemical method, qRT-PCR and/or western blot. ResultsRCOR1 was low-expressed but EDN1 was high-expressed in CP model rats and OGD/R-treated neurons. RCOR1 overexpression ameliorated rat neurobehaviors, alleviated brain pathological conditions, reduced TUNEL-positive cells, decreased the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), increased superoxide dismutase (SOD) level and repressed EDN1 expression in the brains of CP model rats. In neurons, RCOR1 overexpression counteracted OGD/R-induced viability decrease, reduction of the levels of RCOR1, SOD, Bcl-2, caspase-3, p-Akt/Akt and p-GSK-3β/GSK-3β, and elevation of the levels of EDN1, ROS, Bax, and cleaved caspase-3, while EDN1 overexpression did contrarily on these events. Moreover, there was a negative interplay between RCOR1 overexpression and EDN1 overexpression in OGD/R-induced neurons. ConclusionRCOR1 ameliorates neurobehaviors and suppresses neuronal apoptosis and oxidative stress in CP through EDN1 targeting-mediated upregulation of Akt/GSK-3β.

Vitamin D alleviates neuronal injury in cerebral ischemia-reperfusion via enhancing the Nrf2/HO-1 antioxidant pathway to counteract NLRP3-mediated pyroptosis.

Vitamin D supplementation is reported to have anti-inflammatory and neuroprotective effects during cerebral ischemia-reperfusion injury (CIRI), but the protective mechanism has not been fully elucidated. In this study, rats were given prior administrations of 1,25-vitamin D3 (1,25-VitD3) for a week and subjected to 2 hours of middle cerebral artery occlusion (MCAO) followed by 24 hours of reperfusion. Supplementation with 1,25-VitD3 significantly reduced neurological deficit scores and cerebral infarction areas, and increased surviving neurons. Oxygen-glucose deprivation/reoxygenation (OGD/R)-induced rat cortical neuron cells (RN-C) were subjected to 1,25-VitD3 treatment. Administration of 1,25-VitD3 improved cell viability and inhibited lactate dehydrogenase (LDH) activity and cell apoptosis in OGD/R-stimulated RN-C, as assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-h-tetrazolium bromide (MTT) assay, LDH activity assays and TdT-mediated dUTP nick end labeling (TUNEL) staining, respectively. Notably, western blot assay showed that 1,25-VitD3 upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) to alleviate oxidative stress, but reduced proteins and inflammatory cytokines related to NLR pyrin domain containing 3 (NLRP3)-mediated pyroptosis, resulting in decreased pyroptosis and neuroinflammation in vivo and in vitro. Transfection of pcDNA-Nrf2 in RN-C also inhibited pyroptosis and OGD/R-induced cell death whereas breakdown of Nrf2 signals destroyed the protective effect of 1,25-VitD3 on OGD/R-stimulated RN-C. In conclusion, 1,25-VitD3 protects neurons against CIRI through activating the antioxidant Nrf2/HO-1 pathway to restrain NLRP3-mediated pyroptosis.

Piceatannol protects rat neuron cells from oxygen-glucose deprivation reperfusion injury via regulation of GSK-3β/Nrf2 signaling pathway.

To investigate the effect and mechanism of piceatannol on cerebral ischemia-reperfusion injury. The oxygen-glucose deprivation reperfusion (OGD/R) model was constructed in primary cultured suckling rat cortical neuron cells. After 2 h of oxygen-glucose deprivation, the cells were treated with piceatannol for 24 h. The cell survival rate was detected by MTT assay, and the degree of cell damage was detected by intracellular lactate dehydrogenase (LDH) release assay. The activity of superoxide dismutase (SOD) and the content of adenosine triphosphate (ATP) were detected by colorimetric method. The content of reactive oxygen species (ROS) was detected by flow cytometry or observed with inverted fluorescence microscope. The ultrastructure of mitochondria was observed with transmission electron microscopy. Western blotting was used to detect the phosphorylation levels of protein kinase B (AKT) and glycogen synthase kinase (GSK)-3β. Immunofluorescence staining was used to observe the nuclear localization of nuclear factor-erythroid 2-related factor (Nrf) 2. After OGD/R neuron cells were pretreated with Nrf2 inhibitor ML385 for 24 h, the effect of Nrf2 on the improvement of cell activity and antioxidant activity of piceatannol were investigated. Western blotting was used to detect the protein expression levels of Nrf2, heme oxygenase (HO) 1 and NADPH quinone oxidoreductase (NQO) 1. Piceatannol significantly increased the survival rate of OGD/R neurons, decreased LDH release and reactive oxygen species content, increased SOD activity, ameliorated mitochondrial ultrastructural damage, increased mitochondrial membrane potential and ATP level (all P<0.05), increased phosphorylation of AKT and GSK-3β protein, up-regulated the expression of Nrf2, HO-1 and NQO1 protein, increased the nuclear-to-plasma ratio of Nrf2, and promoted the nuclear transfer of Nrf2 (all P<0.05). ML385 could significantly reverse the rescue effect of paclitaxel on the model cells and the regulatory activities of SOD, ROS and LDH (all P<0.05). Piceatannol can regulate Nrf2 by activating GSK-3β signaling pathway, promote its nuclear translocation, exert corresponding antioxidant effect, and protect mitochondrial structure and function in rat neuron cells.

Asymmetric Dimethylarginine Protects Neurons from Oxygen Glucose Deprivation Insult by Modulating Connexin-36 Expression.

Background Asymmetric dimethylarginine (ADMA) is a nonselective nitric oxide synthase inhibitor. ADMA is thought to inhibit the production of nitric oxide (NO) by neurons after oxygen-glucose deprivation (OGD). The gap junction protein Connexin-36 (cx-36) is involved in the pathophysiology of stroke. We investigated whether ADMA could protect neurons from OGD insults by regulating the expression of cx-36. Methods Cultured rat cortical neuronal cells were used. Neurons were treated with OGD with or without ADMA pretreatment. The lactate dehydrogenase (LDH) release rate was used to assess neuronal injury. Intracellular NO levels were determined using 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate. Western blotting was performed to detect cx-36 expression. Results The LDH release rate increased in the supernatant of neurons after the OGD insult, whereas ADMA treatment reduced the LDH release rate. Intracellular NO levels increased following OGD treatment, and this increase was not inhibited by ADMA treatment. Expression of cx-36 was upregulated in neurons under OGD conditions, and treatment with ADMA downregulated the expression of cx-36. Conclusions ADMA protects neurons from OGD insult, and cx-36 downregulation may be a possible pathway involved in ADMA-mediated neuronal protection.

Estrogens still represent an attractive therapeutic approach for Alzheimer's disease.

Estrogens still represent an attractive therapeutic approach for Alzheimer's disease.

Neuronal Exosomes Secreted under Oxygen-Glucose Deprivation/Reperfusion Presenting Differentially Expressed miRNAs and Affecting Neuronal Survival and Neurite Outgrowth.

Ischemia/reperfusion is a key feature of acute ischemic stroke, which causes neuron dysfunction and death. Exosomes, small extracellular vesicles produced by most cell types, are implicated in the mediation of cellular interactions with their environment. Here, we investigated the contents and functions of exosomes from neurons under ischemic reperfusion injury. First, rat cortical primary neuronal cell cultures were placed in an oxygen- and glucose-deprived (OGD) medium, followed by reperfusion in a normoxic conditioned medium (OGD/R) to mimic ischemia/reperfusion in vitro. The neuron-derived exosomes were harvested from the conditioned medium under normoxia and OGD/R. Through next-generation sequencing, exosomal miRNA expression levels in normoxic and OGD/R condition were compared. Their functional activity in terms of neuron viability and quantitative analysis of neurite outgrowth were examined. The expression levels of 45 exosomal miRNAs were significantly different between normoxic and OGD/R conditions. Bioinformatics analysis of dysregulated exosomal miRNAs identified multiple pathways involved in cell survival and death processes and neuronal signaling. Moreover, treatment with exosomes from OGD/R to cultured cortical neurons significantly impaired neuronal cell viability and reduced neurite outgrowth in terms of the number of primary or total neurites as well as length of primary neurites, compared with exosomes from normoxic conditions. miRNA-packed exosomes released by neurons under OGD/R challenge may contribute to post ischemic neuronal injury and provide further understanding of the effect of stressed neurons on neighboring neuronal functions.

Brain-derived neurotropic factor mediates neuroprotection of mesenchymal stem cell-derived extracellular vesicles against severe intraventricular hemorrhage in newborn rats.

Brain‐derived neurotropic factor (BDNF), which is secreted by mesenchymal stem cells (MSCs), protects against severe intraventricular hemorrhage (IVH)‐induced brain injuries. Although the paracrine protective effects of MSCs are mediated primarily by extracellular vesicles (EVs), the therapeutic efficacy of MSC‐derived EVs and the role of the BDNF in the EVs have not been studied. This study aimed to determine whether MSC‐derived EVs attenuate severe IVH‐induced brain injuries, and if so, whether this protection is mediated by BDNF transfer. We compared the therapeutic efficacy of MSCs, MSC‐derived EVs with or without BDNF knockdown, and fibroblast‐derived EVs in vitro in rat cortical neuronal cells challenged with thrombin and in vivo in newborn rats by injecting 200 μL of blood at postnatal day (P) 4 and transplanting 1 × 105 MSCs or 20 μg of EVs at P6. The MSCs and MSC‐derived EVs, but not the EVs derived from BDNF‐knockdown MSCs or fibroblasts, significantly attenuated in vitro thrombin‐induced neuronal cell death and in vivo severe IVH‐induced brain injuries such as increased neuronal cell death, astrogliosis, and inflammatory responses; reduced myelin basic protein and neurogenesis; led to progression of posthemorrhagic hydrocephalus; and impaired behavioral test performance. Our data indicate that MSC‐derived EVs are as effective as parental MSCs in attenuating severe IVH‐induced brain injuries, and this neuroprotection is primarily mediated by BDNF transfer via EVs.

Role of Kv7.2/Kv7.3 and M1 muscarinic receptors in the regulation of neuronal excitability in hiPSC-derived neurons

The Kv7 family of voltage-dependent non-inactivating potassium channels is composed of five members, of which four are expressed in the CNS. Kv7.2, 7.3 and 7.5 are responsible for the M-current, which plays a critical role in the regulation of neuronal excitability. Stimulation of M1 muscarinic acetylcholine receptor, M1 receptor, increases neuronal excitability by suppressing the M-current generated by the Kv7 channel family. The M-current modulation via M1 receptor is well-described in in vitro assays using cell lines and in native rodent tissue. However, this mechanism was not yet reported in human induced pluripotent stem cells (hiPSC) derived neurons. In the present study, we investigated the effects of both agonists and antagonists of Kv7.2/7.3 channel and M1 receptor in hiPSC derived neurons and in primary rat cortical neuronal cells. The role of M1 receptors in the modulation of neuronal excitability could be demonstrated in both rat primary and hiPSC neurons. The M1 receptors agonist, xanomeline, increased neuronal excitability in both rat cortical and the hiPSC neuronal cells. Furthermore, M1 receptor agonist-induced neuronal excitability in vitro was reduced by an agonist of Kv7.2/7.3 in both neuronal cells. These results show that hiPSC derived neurons recreate the modulation of the M-current by the muscarinic receptor in hiPSC neurons similarly to rat native neurons. Thus, hiPSC neurons could be a useful human-based cell assay for characterization of drugs that affect neuronal excitability and/or induce seizure activity by modulation of M1 receptors or inhibition of Kv7 channels.

Microprobe electrode array with individual interconnects through substrate using silicon through-glass via

This paper presents the fabrication and measurements of a vertical out-of-plane microprobe electrode array with individual interconnects through the substrate using a silicon through-glass via (TGV) with transparent properties. The TGV was fabricated using a low-resistance silicon (LRS) via and the glass reflow process. The microprobe structure was formed by multiple deep reactive ion etching and reactive ion etching processes using one-step photolithography and a single etch mask. The height and pitch of the microprobe are designed to be 90 μm and 210 μm, respectively. Cr and Au conductive layers were deposited and patterned on the silicon microprobe structure. A parylene-C thin film was used as an insulating layer and it was etched only at the tip-end through the self-alignment fabrication process using a thick photoresist to expose the conductive part. Each microprobe electrode was independently connected to the backside of the substrate through the silicon TGV. The single via resistance was measured to be 1.26 ± 0.041 Ω. To verify the electrochemical characteristics of the microprobe electrodes with individual interconnects, the steady-state limiting current through the redox reaction was measured by the cyclic voltammetry method for each electrode. The measured steady-state peak current of the microprobe electrode was compared with the theoretical calculation. Further, the electrode impedance was measured and the equivalent circuit analysis was constructed using the Zview impedance modeling software. Experiments were conducted to measure the impedance of 16 microprobe electrodes, and it was confirmed that the impedance did not exceed 1 MΩ at 1 kHz. Then, primary rat cortical neuron cells (DIV 7) were cultured on the fabricated microprobe electrodes and neural spike signals were successfully measured. Also, the light transmittance experiment was conducted to measure the transparency of the TGV structure fabricated through the glass reflow process.

Polyelectrolyte membrane scaffold sustains growth of neuronal cells.

Cell immobilization within nano‐thin polymeric shells can provide an optimal concentration of biological material in a defined space and facilitate its directional growth. Herein, polyelectrolyte membrane scaffolds were constructed using a layer‐by‐layer approach to determine the possibility of promoting improved growth of rat cortical neuronal cells. Membrane presence was confirmed by Fourier transform infrared spectroscopy, Zeta potential, and atomic force and scanning electron microscopy. Scaffold performance toward neuronal cell growth was assessed in vitro during a 14‐day culture. Cell conditions were analyzed immunocytochemically. Furthermore, western blot and real‐time PCR analyses were used to validate the presence of neuronal and glial cells on the scaffolds. We observed that alginate/chitosan, alginate/polylysine, and polyethyleneimine/chitosan scaffolds promote neuronal growth similarly to the control, poly‐d‐lysine/laminin. We conclude that membranes maintaining cell viability, integrity and immobilization in systems supporting neuronal regeneration can be applied in neurological disease or wound healing treatment. © 2018 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 839–850, 2019.

Anandamide Reduces the Toxic Synergism Exerted by Quinolinic Acid and Glutaric Acid in Rat Brain Neuronal Cells

The endocannabinoid system (ECS) regulates several physiological processes in the Central Nervous System, including the modulation of neuronal excitability via activation of cannabinoid receptors (CBr). Both glutaric acid (GA) and quinolinic acid (QUIN) are endogenous metabolites that, under pathological conditions, recruit common toxic mechanisms. A synergistic effect between them has already been demonstrated, supporting potential implications for glutaric acidemia type I (GA I). Here we investigated the possible involvement of a cannabinoid component in the toxic model exerted by QUIN + GA in rat cortical slices and primary neuronal cell cultures. The effects of the CB1 receptor agonist anandamide (AEA), and the fatty acid amide hydrolase inhibitor URB597, were tested on cell viability in cortical brain slices and primary neuronal cultures exposed to QUIN, GA, or QUIN + GA. As a pre-treatment to the QUIN + GA condition, AEA prevented the loss of cell viability in both preparations. URB597 only protected in a moderate manner the cultured neuronal cells against the QUIN + GA-induced damage. The use of the CB1 receptor reverse agonist AM251 in both biological preparations prevented partially the protective effects exerted by AEA, thus suggesting a partial role of CB1 receptors in this toxic model. AEA also prevented the cell damage and apoptotic death induced by the synergic model in cell cultures. Altogether, these findings demonstrate a modulatory role of the ECS on the synergic toxic actions exerted by QUIN + GA, thus providing key information for the understanding of the pathophysiological events occurring in GA I.

Cytotoxic and biological effects of bulk fill composites on rat cortical neuron cells

The aim of this study was to investigate potential cellular responses and biological effects of new generation dental composites on cortical neuron cells in two different exposure times. The study group included five different bulk-fill flow able composites; Surefil SDR Flow, X-tra Base Flow, Venus Bulk Flow, Filtek Bulk Flow and Tetric-Evo Flow. They were filled in Teflon molds (Height: 4 mm, Width: 6 mm) and irradiated for 20 s. Cortical neuron cells were inoculated into 24-well plates. After 80% of the wells were coated, the 3 µm membrane was inserted and dental filling materials were added. The experiment was continued for 24 and 72 h. Cell viability measured by MTT assay test, total antioxidant and total oxidant status were examined using real assay diagnostic kits. The patterns of cell death (apoptosis) were analyzed using annexin V-FITC staining with flow cytometry. Β-defensins were quantitatively assessed by RT-PCR. IL-6, IL-8 and IL-10 cytokines were measured from the supernatants. All composites significantly affected analyses parameters during the exposure durations. Our data provide evidence that all dental materials tested are cytotoxic in acute phase and these effects are induced cellular death after different exposure periods. Significant cytotoxicity was detected in TE, XB, SS, FBF and VBF groups at 24 and 72 h, respectively.

Neuroprotective effects of a novel poly (ADP-ribose) polymerase-1 inhibitor, JPI-289, in hypoxic rat cortical neurons.

Excessive activation of poly (ADP-ribose) polymerase-1 (PARP-1) is known to develop neuronal apoptosis, necrosis and inflammation after ischaemic brain injury. Therefore, PARP-1 inhibition after ischaemic stroke has been attempted in successful animal studies. The purpose of present work was to develop a novel water soluble PARP-1 inhibitor (JPI-289) and explore its neuroprotective effect on ischaemic injury in an in vitro model. The half-life of JPI-289 after intravenous or oral administration in rats was relatively long (1.4-1.5hours) with 65.6% bioavailability. The inhibitor strongly inhibited PARP-1 activity (IC50 =18.5nmol/L) and cellular PAR formation (IC50 =10.7nmol/L) in the nanomolar range. In rat cortical neuronal cells, JPI-289 did not affect cell viability up to 1mmol/L as assayed by Trypan blue staining (TBS) and lactate dehydrogenase (LDH) assay. Treatment of JPI-289 for 2hours after 2hours of oxygen glucose deprived (OGD) rat cortical neuron attenuated PARP activity and restored ATP and NAD+ levels. Apoptosis-associated molecules such as apoptosis inducing factor (AIF), cytochrome C and cleaved caspase-3 were reduced after JPI-289 treatment in the OGD model. The present findings suggest that the novel PARP-1 inhibitor, JPI-289, is a potential neuroprotective agent which could be useful as a treatment for acute ischaemic stroke.

Heme Oxygenase-1 Mediates Neuroprotection Conferred by Argon in Combination with Hypothermia in Neonatal Hypoxia-Ischemia Brain Injury.

Hypoxic-ischemic encephalopathy is a major cause of mortality and disability in the newborn. The authors investigated the protective effects of argon combined with hypothermia on neonatal rat hypoxic-ischemic brain injury. In in vitro studies, rat cortical neuronal cell cultures were challenged by oxygen and glucose deprivation for 90 min and exposed to 70% Ar or N2 with 5% CO2 balanced with O2, at 33°C for 2 h. Neuronal phospho-Akt, heme oxygenase-1 and phospho-glycogen synthase kinase-3β expression, and cell death were assessed. In in vivo studies, neonatal rats were subjected to unilateral common carotid artery ligation followed by hypoxia (8% O2 balanced with N2 and CO2) for 90 min. They were exposed to 70% Ar or N2 balanced with oxygen at 33°, 35°, and 37°C for 2 h. Brain injury was assessed at 24 h or 4 weeks after treatment. In in vitro studies, argon-hypothermia treatment increased phospho-Akt and heme oxygenase-1 expression and significantly reduced the phospho-glycogen synthase kinase-3β Tyr-216 expression, cytochrome C release, and cell death in oxygen-glucose deprivation-exposed cortical neurons. In in vivo studies, argon-hypothermia treatment decreased hypoxia/ischemia-induced brain infarct size (n = 10) and both caspase-3 and nuclear factor-κB activation in the cortex and hippocampus. It also reduced hippocampal astrocyte activation and proliferation. Inhibition of phosphoinositide-3-kinase (PI3K)/Akt pathway through LY294002 attenuated cerebral protection conferred by argon-hypothermia treatment (n = 8). Argon combined with hypothermia provides neuroprotection against cerebral hypoxia-ischemia damage in neonatal rats, which could serve as a new therapeutic strategy against hypoxic-ischemic encephalopathy.

The influence of sterilization on nitrogen-included ultrananocrystalline diamond for biomedical applications

Diamond has shown great potential in different biomedical applications, but the effects of sterilization on its properties have not been investigated. Here, we studied the influence of five sterilization techniques (solvent cleaning, oxygen plasma, UV irradiation, autoclave and hydrogen peroxide) on nitrogen-included ultrananocrystalline diamond. The chemical modification of the diamond surface was evaluated using X-ray photoelectron spectroscopy and water contact angle measurements. Different degrees of surface oxidation and selective sp2 bonded carbon etching were found following all sterilization techniques, resulting in an increase of hydrophilicity. Higher viabilities of in vitro mouse 3T3 fibroblasts and rat cortical neuron cells were observed on oxygen plasma, autoclave and hydrogen peroxide sterilized diamond, which correlated with their higher hydrophilicity. By examination of apatite formation in simulated body fluid, in vivo bioactivity was predicted to be best on those surfaces which have been oxygen plasma treated and lowest on those which have been exposed to UV irradiation. The charge injection properties were also altered by the sterilization process and there appears to be a correlation between these changes and the degree of oxygen termination of the surface. We find that the modification brought by autoclave, oxygen plasma and hydrogen peroxide were most consistent with the use of N-UNCD in biological applications as compared to samples sterilized by solvent cleaning or UV exposure or indeed non-sterilized. A two-step process of sterilization by hydrogen peroxide following oxygen plasma treatment was then suggested. However, the final choice of sterilization technique will depend on the intended end application.

Huwe1 interacts with Gadd45b under oxygen-glucose deprivation and reperfusion injury in primary Rat cortical neuronal cells.

BackgroundGrowth arrest and DNA-damage inducible protein 45 beta (Gadd45b) is serving as a neuronal activity sensor. Brain ischemia induces the expression of Gadd45b, which stimulates recovery after stroke and may play a protective role in cerebral ischemia. However, little is known of the molecular mechanisms of how Gadd45b expression regulated and the down-stream targets in brain ischemia. Here, using an oxygen-glucose deprivation and reperfusion (OGD/R) model, we identified Huwe1/Mule/ARF-BP1, a HECT domain containing ubiquitin ligase, involved in the control of Gadd45b protein level. In this study, we also investigated the role of Huwe1-Gadd45b mediated pathway in BDNF methylation.ResultsWe found that the depletion of Huwe1 by lentivirus shRNA mediated interference significantly increased the expression of Gadd45b and BDNF at 24 h after OGD. Moreover, treatment with Cycloheximide (CHX) inhibited endogenous expression of Gadd45b, and promoted expression of Gadd45b after co-treated with lentivirus shRNA-Huwe1. Inhibition of Gadd45b by lentivirus shRNA decreased the expression levels of brain derived neurotrophic factor (BDNF) and phosphorylated cAMP response element-binding protein (p-CREB) pathway, while inhibition of Huwe1 increased the expression levels of BDNF and p-CREB. Moreover, shRNA-Huwe1 treatment decreased the methylation level of the fifth CpG islands (123 bp apart from BDNF IXa), while shRNA-Gadd45b treatment increased the methylation level of the forth CpG islands (105 bp apart from BDNF IXa).ConclusionsThese findings suggested that Huwe1 involved in the regulation of Gadd45b expression under OGD/R, providing a novel route for neurons following cerebral ischemia-reperfusion injury. It also indicated that the methylation of BDNF IXa was affected by Gadd45b as well as Huwe1 in the OGD/R model.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0178-y) contains supplementary material, which is available to authorized users.

Systematic Asymmetric Synthesis of All Diastereomers of (-)-Talaumidin and Their Neurotrophic Activity.

(-)-Talaumidin (1), a 2,5-biaryl-3,4-dimethyltetrahydrofuran lignan isolated from Aristolochia arcuata Masters, shows significant neurite-outgrowth promotion and neuroprotection in primary cultured rat cortical neurons and in NGF-differentiated PC12 cells. The four stereogenic centers on the tetrahydrofuran moiety in 1 result in the presence of seven diastereomers except for their enantiomers. In order to investigate the stereochemistry-activity relationships of the stereoisomers, the systematic synthesis of all stereoisomers of 1 was accomplished by employing Evans aldol, diastereoselective hydroboration, reductive deoxygenation, and Mitsunobu reactions as key steps. The ability of all of the synthesized stereoisomers to promote neurite-outgrowth in PC12 and neuronal cells was evaluated. All stereoisomers exhibited moderate to potent neurotrophic activities in NGF-differentiated PC12 cells at 30 μM and in primary cultured rat cortical neuronal cells at 0.01 μM. In particular, 1e bearing all cis substituents resulted in the most potent neurite-outgrowth promotion.

Neuroprotective and antioxidant effects of novel benzofuran-2-carboxamide derivatives.

In the present study, we synthesized a series of novel 7-methoxy-N-(substituted phenyl)benzofuran-2-carboxamide derivatives in moderate to good yields and evaluated their neuroprotective and antioxidant activities using primary cultured rat cortical neuronal cells and in vitro cell-free bioassays. Based on our primary screening data with eighteen synthesized derivatives, nine compounds (1a, 1c, 1f, 1i, 1j, 1l, 1p, 1q and 1r) exhibiting considerable protection against the NMDA-induced excitotoxic neuronal cell damage at the concentration of 100 μM were selected for further evaluation. Among the selected derivatives, compound 1f (with -CH3 substitution at R2 position) exhibited the most potent and efficacious neuroprotective action against the NMDA-induced excitotoxicity. Its neuroprotective effect was almost comparable to that of memantine, a well-known NMDA antagonist, at 30 μM concentration. In addition to 1f, compound 1j (with -OH substitution at R3 position) also showed marked anti-excitotoxic effects at both 100 and 300 μM concentrations. These findings suggest that -CH3 substitution at R2 position and, to a lesser degree, -OH substitution at R3 position may be important for exhibiting neuroprotective action against excitotoxic damage. Compound 1j was also found to scavenge 1,1-diphenyl-2-picrylhydrazyl radicals and inhibit in vitro lipid peroxidation in rat brain homogenate in moderate and appreciable degrees. Taken together, our structure-activity relationship studies suggest that the compound with -CH3 substitution at R2 and -OH substitution at R3 positions of the benzofuran moiety might serve as the lead exhibiting potent anti-excitotoxic, ROS scavenging, and antioxidant activities. Further synthesis and evaluation will be necessary to confirm this possibility.

Polydimethylsiloxane Microstencils Molded on 3-D-Printed Templates

Microstencils have been utilized in biomedical engineering to pattern cell cultures, engineer tissues, and pattern conductive materials in microelectronics for the measurement of bioelectric activity. However, fabricating these microstencils can be considerably time consuming, expensive, and cleanroom processing or laser micromachining intensive. We present microfabrication strategies for producing stencils rapidly and cost effectively, with minimal use of cleanroom facilities, ideal for prototyping or applications where microfabrication costs need to be conserved. The process utilizes 3-D-printed templates as master structures from, which polydimethylsiloxane (PDMS) microstencils are molded. The entire process, from concept to completed stencil, requires approximately one day; however, the majority of this time is budgeted for PDMS curing cycles, so it requires only ~1-2 person hours to complete. These microstencils were used to pattern metal traces ~160-1000-μm wide, on three commonly used BioMEMS substrate materials- to pattern rat cortical neuronal cell cultures with radii between ~300 and 1000 μm-and to pattern organic materials. With the advancement of 3-D-printing technologies, we anticipate that our presented processes will improve in resolution and gain a greater cost advantage over traditional microstencilling methods.

Downregulation of dopamine D₁ receptors and increased neuronal apoptosis upon ethanol and PTZ exposure in prenatal rat cortical and hippocampal neurons.

The objective of this study was to evaluate the effect of ethanol and pentylenetetrazol (PTZ) on the expression of dopamine receptors (D1R) and to observe the apoptotic neurodegeneration in prenatal rat cortical and hippocampal neurons at gestational days (GD) 17.5. In the present study, ethanol (100 mM) and PTZ (15 mM) were exposed to the prenatal rat cortical and hippocampal neuronal cell cultures for 1 h. For mRNA RT-PCR and for protein Western blot analysis was done to elucidate D1R, Bax, Bak, Bcl-2 and cleaved caspase-3 expression upon ethanol and PTZ exposure in neuronal cell cultures. Furthermore, ethanol and PTZ-induced apoptotic neurodegeneration was also observed using TUNEL staining and propidium iodide (PI) used as counter stain under confocal microscopy. The results of present study showed that ethanol and PTZ exposure significantly decreased D1R expression and induced neuronal death by significantly increasing the expression of pro-apoptotic Bax, Bak and decreasing anti-apoptotic protein Bcl-2 leading to the apoptosis by increasing cleaved caspase-3 expression in cortical and hippocampal primary neuronal cell cultures. Our findings indicated that ethanol and PTZ exposure to the prenatal neurons showed not only downregulation of D1R but also causes neuronal apoptosis in the developing rat brain. Further, this explains the possibility of higher risk of developmental disturbances and malformations during early developmental stage.