Cortical Neuronal Cell Cultures Research Articles

Protective Effects of Cannabidiol (CBD) against Qxidative Stress, but Not Excitotoxic-Related Neuronal Cell Damage-An In Vitro Study.

Cannabidiol (CBD) appears to possess some neuroprotective properties, but experimental data are still inconsistent. Therefore, this in vitro study aimed to compare the effects of CBD in a wide range of concentrations on oxidative stress and excitotoxic-related cell damage. Results showed that low concentrations of CBD ameliorated the H2O2-evoked cell damage of primary cortical neuronal cell culture. However, higher concentrations of CBD alone (5-25 μM) decreased the viability of cortical neurons in a concentration-dependent manner and aggravated the toxic effects of hydrogen peroxide (H2O2). Neuroprotection mediated by CBD in primary neurons against H2O2 was not associated with a direct influence on ROS production nor inhibition of caspase-3, but we found protective effects of CBD at the level of mitochondrial membrane potential and DNA fragmentation. However, CBD had no protective effect on the glutamate-induced cell damage of cortical neurons, and in higher concentrations, it enhanced the toxic effects of this cell-damaging factor. Likewise, CBD, depending on its concentration, at least did not affect or even enhance cortical cellular damage exposed to oxygen-glucose deprivation (OGD). Finally, we showed that CBD in submicromolar or low micromolar concentrations significantly protected human neuronal-like SH-SY5Y cells against H2O2- and 6-hydroxydopamine (6-OHDA)-induced cell damage. Our data indicate that CBD has a dual effect on oxidative stress-induced neuronal death-in low concentrations, it is neuroprotective, but in higher ones, it may display neurotoxic activity. On the other hand, in excitotoxic-related models, CBD was ineffective or enhanced cell damage. Our data support the notion that the neuroprotective effects of CBD strongly depend on its concentration and experimental model of neuronal death.

Redox Protein Thioredoxin Mediates Neurite Outgrowth in Primary Cultured Mouse Cerebral Cortical Neurons

Thioredoxin system plays an important role in maintaining the cellular redox balance. Recent evidence suggests that thioredoxin (Trx) system may promote cell survival and neuroprotection. In this study, we explored the role of thioredoxin system in neuronal differentiation using a primary mouse cortical neuronal cell culture. First, Trx and Trx reductase (TrxR) protein levels were analyzed in cultured neurons from 1 to 32 days in vitro (DIV). The result showed that Trx and TrxR protein levels time-dependently increased in the neuron cell culture from 1 to 18 DIV. To establish the role of Trx in neuronal differentiation, Trx gene expression was knockdown in cultured neurons using Trx sgRNA CRISPR/Cas9 technology. Treatment with CRISPR/Cas9/Trx sgRNA decreased Trx protein levels and caused a reduction in dendritic outgrowth and branching of cultured neurons. Then, primary cortical neurons were treated with the Trx inhibitor PX12 to block Trx reducing activity. Treatment with PX12 also reduced dendritic outgrowth and branching. Furthermore, PX12 treatment reduced the ratio of phosphorylated cyclic AMP response element-binding protein (CREB)/total CREB protein levels. To investigate whether CREB phosphorylation is redox regulated, SH-SY5Y cells were treated with H2O2, which reduced phosphorylated CREB protein levels and increased CREB thiol oxidation. However, treatment with CB3, a Trx-mimetic tripeptide, rescued H2O2-decreased CREB phosphorylation. Our results suggest that Trx regulates neuronal differentiation and maturation of primary mouse cortical neurons by targeting CREB neurotrophic pathway. Trx may regulate CREB activation by maintaining the cellular redox balance.

Klotho increases antioxidant defenses in astrocytes and ubiquitin–proteasome activity in neurons

Klotho is an antiaging protein, and its levels decline with age and chronic stress. The exogenous administration of Klotho can enhance cognitive performance in mice and negatively modulate the Insulin/IGF1/PI3K/AKT pathway in terms of metabolism. In humans, insulin sensitivity is a hallmark of healthy longevity. Therefore, this study aimed to determine if exogenous Klotho, when added to neuronal and astrocytic cell cultures, could reduce the phosphorylation levels of certain insulin signaling effectors and enhance antioxidant strategies in these cells. Primary cell cultures of cortical astrocytes and neurons from mice were exposed to 1 nM Klotho for 24 h, with or without glucose. Klotho decreased pAKT and mTOR levels. However, in astrocytes, Klotho increased FOXO-3a activity and catalase levels, shielding them from intermediate oxidative stress. In neurons, Klotho did not alter FOXO-3 phosphorylation levels but increased proteasome activity, maintaining lower levels of PFKFB3. This study offers new insights into the roles of Klotho in regulating energy metabolism and the redox state in the brain.

Acai Berry (Euterpe sp.) Extracts Are Neuroprotective against L-Glutamate-Induced Toxicity by Limiting Mitochondrial Dysfunction and Cellular Redox Stress.

Aberrant accumulation of the neurotransmitter L-glutamate (L-Glu) has been implicated as a mechanism of neurodegeneration, and the release of L-Glu after stroke onset leads to a toxicity cascade that results in neuronal death. The acai berry (Euterpe oleracea) is a potential dietary nutraceutical. The aim of this research was to investigate the neuroprotective effects of acai berry aqueous and ethanolic extracts to reduce the neurotoxicity to neuronal cells triggered by L-Glu application. L-Glu and acai berry effects on cell viability were quantified using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, and effects on cellular bioenergetics were assessed via quantitation of the levels of cellular ATP, mitochondrial membrane potential (MMP), and production of reactive oxygen species (ROS) in neuroblastoma cells. Cell viability was also evaluated in human cortical neuronal progenitor cell culture after L-Glu or/and acai berry application. In isolated cells, activated currents using patch-clamping were employed to determine whether L-Glu neurotoxicity was mediated by ionotropic L-Glu-receptors (iGluRs). L-Glu caused a significant reduction in cell viability, ATP, and MMP levels and increased ROS production. The co-application of both acai berry extracts with L-Glu provided neuroprotection against L-Glu with sustained cell viability, decreased LDH production, restored ATP and MMP levels, and reduced ROS levels. Whole-cell patch-clamp recordings showed that L-Glu toxicity is not mediated by the activation of iGluRs in neuroblastoma cells. Fractionation and analysis of acai berry extracts with liquid chromatography-mass spectrometry identified several phytochemical antioxidants that may have provided neuroprotective effects. In summary, the acai berry contains nutraceuticals with antioxidant activity that may be a beneficial dietary component to limit pathological deficits triggered by excessive L-Glu accumulations.

Dopamine and serotonin alterations by Hizikia fusiformis extracts under in vitro cortical primary neuronal cell cultures.

Hizikia fusiformis (HF) is a class of brown seaweeds whose active ingredients exert central nervous system protective effects, such as neuroprotection; however, the underlying mechanisms remain unknown. Given that dopamine (DA) and serotonin (5HT) are two major neurotransmitters involved in various psychiatric disorders and neuronal growth in early neurodevelopmental processes, we investigated whether HF extract could modulate the molecular expression associated with DA and 5HT transmission as well as the structural formation of neurons. In vitro cell cultures were prepared from cerebral cortical neurons obtained from CD-1 mice on embryonic day 14. Cultured cells were treated with 0.1, 1.0, or 10.0 μg/mL of HT extract for 24 h, followed by fluorescence immunostaining for DA and 5HT-related receptors and transporters and some neuronal structural formation-associated molecules. HF extract dose-dependently upregulated the expression levels of selective DA and 5HT receptors, and downregulated the levels of DA and 5HT transporters. Moreover, HF extract increased the neurofilament light chain expression. These results suggest that HF may modulate DA and 5HT transmission, thereby affecting neurodevelopment.

Neuroprotective Effect of Clobenpropit against Lipopolysaccharide-Induced Cognitive Deficits via Attenuating Neuroinflammation and Enhancing Mitochondrial Functions in Mice.

Clobenpropit (CLO), an antagonist on histamine H3 receptors (HH3R), has been shown to protect NMDA-induced neuronal necrosis in cortical neuronal cell culture from rats. In this work, we explored its potential on lipopolysaccharide (LPS)-induced memory deficits, neuroinflammation, and mitochondrial dysfunction in mice. CLO (1 and 3 mg/kg, p.o.) was treated continually for 30 days, and neurotoxicity was induced by four doses of LPS (250 µg/kg, i.p.). The radial arm maze (RAM) was used to access memory behaviors. After the REM test, brain tissue was collected from each mouse to estimate pro-inflammatory cytokines (TNFα and IL6), anti-inflammatory cytokines (TGF-β1 and IL-10), cyclooxygenase-2 (COX 2), and mitochondrial respiratory chain complex (MRCC- I, II and IV) enzymes. CLO treatment reversed the LPS-induced behavioral deficits by a significant reduction in time taken to consume all five bites (TTB), working memory error (WME), and reference memory error (REM) in the REM test. Regarding neuroinflammation, it attenuated the release of COX, TNF-α, and IL-6, and augmented TGF-β1 and IL-10 levels in the brain. Reversal of LPS-induced brain MRCC (I, II, and IV) levels also resulted with CLO treatment. From these findings, CLO promises neuroprotection against LPS-induced cognitive deficits by ameliorating neuroinflammation and restoring the MRCC enzymes in mice.

β-Asarone suppresses TNF-α expression through DNA methylation and c-Jun-mediated transcription modulation in scratch-injured neuronal cells.

This study aimed to investigate the role and possible mechanism of β-asarone in regulating neuronal apoptosis and axonal regeneration. A scratch injury was applied to cell cultures of mouse primary cortical neurons to mimic neuronal injury. The neuronal apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelingstaining and western blot analysis of apoptosis-related proteins. The axonal regeneration was assessed by immunofluorescent staining of β-tubulin III and western blot analysis of axonal markers. In the results, β-asarone inhibited neuronal apoptosis and promoted axonal regeneration by suppressing tumor necrosis factor-α (TNF-α) expression in scratch-injured mouse neuronal cells. Research investigating the molecular mechanisms by which β-asarone inhibited TNF-α expression showed that, on the one hand, β-asarone suppressed the JNK/c-Jun pathway and thus transcriptionally inhibited TNF-α expression; on the other hand, β-asarone induced expression of UHRF1 that recruited DNMT1 to induce TNF-α promoter methylation and subsequently decreased the messenger RNA expression of TNF-α. In conclusion, β-asarone suppresses TNF-α expression through DNA methylation and c-Jun-mediated transcription modulation in scratch-injured neuronal cells.

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.

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.

In Vitro and In Vivo Analysis of the Effects of 3,5-DMA and Its Metabolites in Neural Oxidative Stress and Neurodevelopmental Toxicity.

3,5-Dimethylaniline (3,5-DMA), a monocyclic aromatic amine, is widely present in a spectrum of sources including tobacco, dyes, combustion products, and suspended particulates. 3,5-DMA and its metabolites form superoxides, resulting in apoptosis or oncogenesis. Data of a direct effect of 3,5-DMA on the nervous system, especially the developing brain, are lacking. Therefore, we investigated the effects of 3,5-DMA and its metabolites on fetal neurite growth and brain development using in vitro cell cultures of primary cortical neurons to observe whether these compounds caused neuronal cytotoxicity and affected neurite structural development. With increasing concentrations of 3,5-DMA (10, 50, 100, 500, 1000 μM) and its major metabolite 5-dimethylaminophenol (3,5-DMAP) (10, 50, 100, 500, 1000 μM), reactive oxygen species (ROS), cytotoxicity, and DNA damage increased significantly in the cells and dendritic arborization decreased. The addition of 5 mM N-acetylcysteine, an ROS scavenger, reduced ROS in the cells and alleviated the neuronal damage. In vivo studies in Sprague Dawley pregnant rats suggested that exposure to 3,5-DMA (10, 30, 60, 100 mg/kg/day) subcutaneously from GD15 to GD17 led to fetal cerebral cortex thinning. BrdU labeling showed that 3,5-DMA reduced the number and generation of cortical cells. To detect the laminar position of newly generated neurons, cortex layer markers such as Satb2, Ctip2, and Tbr1 were used. 3,5-DMA perturbed the cortical layer distribution in developing fetal rats. In summary, this is the first study to provide evidence for 3,5-DMA and its metabolites causing anomalies of the fetal central nervous system development through ROS production.

New evidences for a role of mGluR7 in astrocyte survival: Possible implications for neuroprotection

A specific activation of metabotropic glutamate receptor 7 (mGluR7) has been shown to be neuroprotective in various models of neuronal cell damage, however, its role in glia cell survival has not been studied, yet. Thus, we performed comparative experiments estimating protective effects of the mGluR7 allosteric agonist AMN082 in glia, neuronal and neuronal-glia cell cultures against various harmful stimuli. First, the transcript levels of mGluR7 and other subtypes of group II and III mGluRs in cortical neuronal, neuronal-glia and glia cell cultures have been measured by qPCR method. Next, we demonstrated that AMN082 with similar efficiency attenuated the glia cell damage evoked by staurosporine (St) and doxorubicin (Dox). The AMN082-mediated glioprotection was mGluR7-dependent and associated with decreased DNA fragmentation without involvement of caspase-3 inhibition. Moreover, the inhibitors of PI3K/Akt and MAPK/ERK1/2 pathways blocked the protective effect of AMN082. In neuronal and neuronal-glia cell cultures in the model of glutamate (Glu)- but not St-evoked cell damage, we showed a significant glia contribution to mGluR7-mediated neuroprotection. Finally, by using glia and neuronal cells derived from mGluR7+/+ and mGluR7−/− mice we demonstrated a higher cell-damaging effect of St and Dox in mGluR7-deficient glia but not in neurons (cerebellar granule cells). Our present data showed for the first time a glioprotective potential of AMN082 underlain by mechanisms involving the activation of PI3K/Akt and MAPK/ERK1/2 pathways and pro-survival role of mGluR7 in glia cells. These findings together with the confirmed neuroprotective properties of AMN082 justify further research on mGluR7-targeted therapies for various CNS disorders.

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.

Argon protects against hypoxic-ischemic brain injury in neonatal rats through activation of nuclear factor (erythroid-derived 2)-like 2.

Perinatal hypoxic ischaemic encephalopathy (HIE) has a high mortality rate with neuropsychological impairment. This study investigated the neuroprotective effects of argon against neonatal hypoxic-ischaemic brain injury.In vitro cortical neuronal cell cultures derived from rat foetuses were subjected to an oxygen and glucose deprivation (OGD) challenge for 90 minutes and then exposed to 70% argon or nitrogen with 5% carbon dioxide and balanced with oxygen for 2 hours.In vivo, seven-day-old rats were subjected to unilateral common carotid artery ligation followed by hypoxic (8% oxygen balanced with nitrogen) insult for 90 minutes. They were exposed to 70% argon or nitrogen balanced with oxygen for 2 hours. In vitro, argon treatment of cortical neuronal cultures resulted in a significant increase of p-mTOR and Nuclear factor (erythroid-derived 2)-like 2(Nrf2) and protection against OGD challenge. Inhibition of m-TOR through Rapamycin or Nrf2 through siRNA abolished argon-mediated cyto-protection. In vivo, argon exposure significantly enhanced Nrf2 and its down-stream effector NAD(P)H Dehydrogenase, Quinone 1(NQO1) and superoxide dismutase 1(SOD1). Oxidative stress, neuroinflammation and neuronal cell death were significantly decreased and brain infarction was markedly reduced. Blocking PI-3K through wortmannin or ERK1/2 through U0126 attenuated argon-mediated neuroprotection.These data provide a new molecular mechanism for the potential application of argon as a neuroprotectant in HIE.

Search of Neurotrophin-mimic Natural Products for Prevention and Treatment of Neurodegenerative Disease

As part of our continuing studies on neurotrophin-mimic active compounds in natural products, we investigated the chemical constituents of the pericarps of Illicium jiadifengpi and the roots of Indonesian ginger Zingiber purpureum, resulting in the isolation of new seco-prezizaane-type sesquiterpenoid 1 and phenylbutenoid dimer 3-4 and two new curcuminoids 5-6. The MeOH extract of I. jiadifengpi was fractionated, leading to the isolation of compound 1. Compound 1 significantly enhanced neurite outgrowth in primary cell cultures of fetal rat cortical neurons. It is noteworthy that compound 1 has potential significantly to promote differentiation of multipotent neural stem cell line (MEB5 cells) into neurons. Additionally, we investigated the MeOH extract of the root of Bangle (Z. purpureum) that exhibited neuritogenesis activity in PC12 cells at 25 μg/mL, resulting in the isolation of neurotrophic phenylbutenoid dimers 3-4 and new compounds 5-6. Compounds 3 and 4 were found not only significantly to induce neurite sprouting of PC12 cells but also to increase the neurite length and number of neurites in primary cultured rat cortical neurons, and also showed protective activity against cell death caused by deprivation of serum. Furthermore, chronic treatment with these compounds enhanced hippocampal neurogenesis in dementia model olfactory bulbectomized (OBX) mice. Compounds 5 and 6 had significant NGF-potentiating effects on PC12 cells whereas compound 5 enhanced prevention of amyloid β (Aβ) 42 aggregation.

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.

Neuroprotective effects of erythromycin on cerebral ischemia reperfusion-injury and cell viability after oxygen-glucose deprivation in cultured neuronal cells

This study aims to determine if erythromycin has neuroprotective effects against transient ischemia and oxygen-glucose deprivation (OGD) in cultured neuronal cells. Sprague-Dawley rats were subjected to middle cerebral artery occlusion for 90min, followed by reperfusion. The animals received a subcutaneous single injection of erythromycin lactobionate (EM, 50mg/kg) or vehicle immediately after ischemia. Infarct volume, edema index, and neurological performance were evaluated at 24 and 72h after reperfusion. Immunohistochemical analyses for oxidative stress (4-HNE, 8-OHdG) and inflammation (Iba-1, TNF-α) were conducted in the cortex at 24h. Primary cortical neuronal cell cultures were prepared from the cerebral cortices of the animals and then subjected to OGD for 3h. Ten or 100μM EM was added before OGD to determine the effect of EM on cell viability after OGD. EM significantly reduced infarct volume (p<0.01) and edema volume (p<0.05) and improved neurological deficit scores (p<0.05) at 24 and 72h. EM significantly suppressed the accumulation of 4-HNE (p<0.01) and 8-OHdG (p<0.01) and markedly reduced Iba-1 (p<0.01) and TNF-α expression (p<0.01). Treatment with 100M EM in vitro significantly reduced cell death after OGD. EM reduces neuronal damage following cerebral ischemia and OGD and may have antioxidant and anti-inflammatory effects.

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.

Effects of Dizocilpine (MK-801) via Up-modulation of N-methyl-D-aspartate (NMDA) Receptors on Hypoxic-Ischemic Brain Injury in Neonatal Rats

Objective : Several studies have demonstrated the neuroprotective effects of (+)-MK-801 hydrogen maleate (dizocilpine), in various animal models of hypoxic-ischemic (HI) brain injury. However limited data are available on the neonatal model of HI brain injury. The aim of the present study was to investigate the effects of dizocilpine and its mechanisms associated with NMDARs expression in neonatal rat model of HI brain injury. Methods : In in vivo model, 7d-old rat pups underwent permanent unilateral carotid ligation. The animals were divided into six groups: N, normoxia; H, hypoxia without operation; HS, hypoxia with Sham operation; HO, hypoxia with operation; HV, HO treated with vehicle; HD, HO treated with dizocilpine. Dizocilpine (10 mg/kg) was administered intracerebrally to the rats 30 min before HI brain injury. Rat pups were exposed to hypoxia by placing them for 2 hours in hypoxic incubator (92% N2, 8% O2). In in vitro model, embryonic cortical neuronal cell cultures (from SD rats of embryonic days of 18) were done. The normoxia (N) group was prepared in 5% CO2 incubators. The hypoxia (H), and hypoxia treated with dizocilpine (HD) groups were placed in 1% O2 incubators (94% N2, 5% CO2) for 16 hours. In order to estimation of cell viability and growth, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was done. The degree of neuronal death was evaluated by morphometric method and the protein expression of each NMDARs was quantified by Real Time-PCR and Western blot. Results : Both in the in vitro and in vivo models, the expressions of NMDAR subunits were lower in the hypoxia group than in the normoxia group, whereas they increased in the hypoxia treated with dizocilpine group compared to the hypoxia group. In vitro model, however, the expressions of NR1, NR2A mRNAs decreased in the H group when compared to the N group, whereas they increased a little in the HD group when compared to the H group. Conclusion : Dizocilpine was modulated the degeneration of neuronal cell death in neonatal rat model of HI by preservation of NR expression.

Taurine Protection against Endoplasmic Reticulum Stress in an Animal Stroke Model of Cerebral Artery Occlusion and Stroke Related conditions in Primary Neuronal Cell Culture

Taurine has been shown to provide protection against neurological diseases, such as Huntington's and stroke. We have shown that taurine can provide effective protection against Endoplasmic Reticulum (ER) stress induced by excitotoxicity or oxidative stress in PC12 cell line or primary neuronal cell cultures. In this study, we employed hypoxia/reoxygenation conditions for primary cortical neuronal cell cultures as well as the model of rat focal Middle Cerebral Artery Occlusion (MCAO). Using taurine greatly increased cell viability in primary neuronal cell culture and decreased the infarct area of sections at 6mm from 47.42±9.86% to 26.76 ± 6.91% in the MCAO model. Furthermore levels of the ER stress protein markers GRP78, Caspase12, CHOP and p‐IRE‐1 which were markedly increased in both the in vitro and in vivo models, significantly declined after taurine administration, suggesting that taurine may exert neuroprotection functions in both models. Moreover, taurine could downregulate the ratio of cleaved ATF6 and full length ATF6 in both models. In the animal model of stroke, taurine induced an upregulation of the Bcl‐2/Bax ratio and downregulation of Caspase‐3 protein activity indicating that it attenuates apoptosis in the core of the ischemic infarct. Our results show not only taurine elicits neuroprotection through the activation of the ATF6 and the IRE1 pathways, but also it can reduce apoptosis in these modelsGrant Funding Source: 09KW‐11, Department of Health, State of Florida

In vitro MS‐based proteomic analysis and absolute quantification of neuronal‐glial injury biomarkers in cell culture system

MS-based proteomics has been the method of choice for biomarker discovery in the field of traumatic brain injury (TBI). Due to its high sensitivity and specificity, MS is now being explored for biomarker quantitative validation in tissue and biofluids. In this study, we demonstrate the use of MS in both qualitative protein identification and targeted detection of acute TBI biomarkers released from degenerating cultured rat cortical mixed neuronal cells, mimicking intracellular fluid in the central nervous system after TBI. Calpain activation was induced by cell treatment with maitotoxin (MTX), a known calcium channel opener. Separate plates of mixed neuronal-glial culture were subjected to excitotoxin N-methyl-D-aspartate (NMDA) and apoptotic inducer staurosporine. Acute TBI biomarkers, GFAP and UCH-L1, were first detected and assessed in the culture media by Western blot. The cell-conditioned media were then trypsinized and subjected to bottom up proteomic analysis. GFAP was readily detected by data-dependent scanning but not UCH-L1. As a proof-of-principle study, rat glia-enriched cell cultures treated with MTX were used to investigate the time-dependent release of GFAP breakdown product by Western blot and for isotope dilution MS absolute quantitation method development. Absolute quantitation of the GFAP release was conducted using the three cortical mixed neuronal cell cultures treated with different agents. Other differentially expressed proteins identified in the glial-enriched and cortical mixed neuronal cell culture models were further analyzed by bioinformatic tools. In summary, this study demonstrates the use of MS in both protein identification and targeted quantitation of acute TBI biomarkers and is the preliminary step toward development of TBI biomarker validation by targeted MS.