Brain Cell Cultures Research Articles

Adenosine‐to‐Inosine RNA Editing in Mouse and Human Brain Proteomes

Proteogenomics is based on the use of customized genome or RNA sequencing databases for interrogation of shotgun proteomics data in search for proteome-level evidence of genome variations or RNA editing. In this work, the products of adenosine-to-inosine RNA editing in human and murine brain proteomes are identified using publicly available brain proteome LC-MS/MS datasets and an RNA editome database compiled from several sources. After filtering of false-positive results, 20 and 37 sites of editing in proteins belonging to 14 and 32 genes are identified for murine and human brain proteomes, respectively. Eight sites of editing identified with high spectral counts overlapped between human and mouse brain samples. Some of these sites have been previously reported using orthogonal methods, such as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors, CYFIP2, coatomer alpha. Also, differential editing between neurons and microglia is demonstrated in this work for some of the proteins from primary murine brain cell cultures. Because many edited sites are still not characterized functionally at the protein level, the results provide a necessary background for their further analysis in normal and diseased cells and tissues using targeted proteomic approaches.

The Orphan Cytokine Receptor CRLF3 Emerged With the Origin of the Nervous System and Is a Neuroprotective Erythropoietin Receptor in Locusts.

The orphan cytokine receptor-like factor 3 (CRLF3) was identified as a neuroprotective erythropoietin receptor in locust neurons and emerged with the evolution of the eumetazoan nervous system. Human CRLF3 belongs to class I helical cytokine receptors that mediate pleiotropic cellular reactions to injury and diverse physiological challenges. It is expressed in various tissues including the central nervous system but its ligand remains unidentified. A CRLF3 ortholog in the holometabolous beetle Tribolium castaneum was recently shown to induce anti-apoptotic mechanisms upon stimulation with human recombinant erythropoietin. To test the hypothesis that CRLF3 represents an ancient cell-protective receptor for erythropoietin-like cytokines, we investigated its presence across metazoan species. Furthermore, we examined CRLF3 expression and function in the hemimetabolous insect Locusta migratoria. Phylogenetic analysis of CRLF3 sequences indicated that CRLF3 is absent in Porifera, Placozoa and Ctenophora, all lacking the traditional nervous system. However, it is present in all major eumetazoan groups ranging from cnidarians over protostomians to mammals. The CRLF3 sequence is highly conserved and abundant amongst vertebrates. In contrast, relatively few invertebrates express CRLF3 and these sequences show greater variability, suggesting frequent loss due to low functional importance. In L. migratoria, we identified the transcript Lm-crlf3 by RACE-PCR and detected its expression in locust brain, skeletal muscle and hemocytes. These findings correspond to the ubiquitous expression of crlf3 in mammalian tissues. We demonstrate that the sole addition of double-stranded RNA to the culture medium (called soaking RNA interference) specifically interferes with protein expression in locust primary brain cell cultures. This technique was used to knock down Lm-crlf3 expression and to abolish its physiological function. We confirmed that recombinant human erythropoietin rescues locust brain neurons from hypoxia-induced apoptosis and showed that this neuroprotective effect is absent after knocking down Lm-crlf3. Our results affirm the erythropoietin-induced neuroprotective function of CRLF3 in a second insect species from a different taxonomic group. They suggest that the phylogenetically conserved CRLF3 receptor may function as a cell protective receptor for erythropoietin or a structurally related cytokine also in other animals including vertebrate and mammalian species.

Real Time Videomicroscopy and Semiautomated Analysis of Brain Cell Culture Models of Trinucleotide Repeat Expansion Diseases.

Proper brain function requires the coordinated and intricate interaction between neuronal and glial cells. Like many other neurological conditions, trinucleotide repeat expansion disorders are likely initiated by the synergistic combination of abnormalities hitting different brain cell types, which ultimately disrupt brain function and lead to the onset of neurological symptoms. Understanding how trinucleotide repeat expansions affect the phenotypes and physiology of neurons and glia is fundamental to improve our understanding of disease mechanisms in the brain and shape the design of future therapeutic interventions.Here we describe a protocol for semiautomated videomicroscopy analysis of cultured brain cells, maintained under suitable and controlled conditions. Through real-time monitoring of basic cell phenotypes (such as proliferation, cell morphology, differentiation, and migration) this method provides an accurate primary assessment of the impact of the repeat expansion on the physiology of neurons and glia. The versatility of the system, the automated image acquisition and the semiautomated processing of the data collected allow rapid phenotypic analysis of individual cell types, as well as the investigation of cell-cell interactions. The stability of the acquisition system provides reproducible and robust results. The raw data can be easily exported to other software to perform more sophisticated imaging analysis and statistical tests. In summary, the methods described offer versatile, reproducible, and time-effective means to dissect the impact of the repeat expansion on different brain cell types and on intercellular interactions.

Exploiting nanogroove-induced cell culture anisotropy to advance in vitro brain models

A new generation of in vitro human brain models is vital to surpass the limitations of current cell culture platforms and animal cell lines in studying brain function and diseases. Brain-on-chip technology can generate well-defined and reproducible platforms to control the cellular microenvironment for in vivo-like, organized brain cell cultures. Previously, the authors investigated differentiation and network organization of the neuroblastoma SH-SY5Y cell line on nanogrooved substrates, showing that nanogroove guidance of neuronal outgrowths is dependent on nanogroove dimensions. Further, increased orientation of neurites was positively correlated to the differentiation of SH-SY5Y cells. However, as mimicking brain structure alone is insufficient, here, the function of the neuronal cell network as dependent on surface topography and material stiffness is investigated. A generalized replication protocol was developed to create similar nanogrooved patterns in cell culture substrates from different materials, specifically polydimethylsiloxane (PDMS) and Ostemer. Experiments using calcium imaging, where calcium fluxes across membranes are visualized as an indication of action potentials in neuronal cells, were performed with differentiated SH-SY5Y cells and human induced pluripotent stem cell-derived neuronal cells (hiPSCNs) on flat versus nanogrooved substrates to study the network function. Calcium live-imaging was performed and results for experiments with SH-SY5Y cells and hiPSCNs showed that nanogrooved PDMS substrates trended toward increased cellular activity and neuronal cell network connectivity. For future investigation of compatible substrate materials in combination with the effect of material stiffness on the cells, nanogrooved Ostemer substrates were demonstrated to faithfully replicate for use in neuronal cell cultures using nanogrooved substrates. First experiments into the neuronal cell function using stem cells described here aid toward elucidating the effect of nanotopographical and mechanical properties and their benefits toward advancing in vitro neuronal cell models both in form and function. Overall, the results indicate, in conjunction with the previous findings on neuronal outgrowth guidance, that anisotropy as introduced by nanogrooved substrates can have a controllable and potentially beneficial influence on neuronal cell cultures.

Model-based estimation of lowest observed effect concentration from replicate experiments to identify potential biomarkers of in vitro neurotoxicity.

A paradigm shift is occurring in toxicology following the report of the National Research Council of the USA National Academies entitled "Toxicity testing in the 21st Century: a vision and strategy". This new vision encourages the use of in vitro and in silico models for toxicity testing. In the goal to identify new reliable markers of toxicity, the responsiveness of different genes to various drugs (amiodarone: 0.312-2.5[Formula: see text]; cyclosporine A: 0.25-2[Formula: see text]; chlorpromazine: 0.625-10[Formula: see text]; diazepam: 1-8[Formula: see text]; carbamazepine: 6.25-50[Formula: see text]) is studied in 3D aggregate brain cell cultures. Genes' responsiveness is quantified and ranked according to the Lowest Observed Effect Concentration (LOEC), which is estimated by reverse regression under a log-logistic model assumption. In contrast to approaches where LOEC is identified by the first observed concentration level at which the response is significantly different from a control, the model-based approach allows a principled estimation of the LOEC and of its uncertainty. The Box-Cox transform both sides approach is adopted to deal with heteroscedastic and/or non-normal residuals, while estimates from repeated experiments are summarized by a meta-analytic approach. Different inferential procedures to estimate the Box-Cox coefficient, and to obtain confidence intervals for the log-logistic curve parameters and the LOEC, are explored. A simulation study is performed to compare coverage properties and estimation errors for each approach. Application to the toxicological data identifies the genes Cort, Bdnf, and Nov as good candidates for in vitro biomarkers of toxicity.

Mimicking the Brain Extracellular Matrix in Vitro: A Review of Current Methodologies and Challenges

AbstractThe brain's extracellular matrix (ECM), a 3‐dimensional macromolecular network that supports cell growth and viability, has crucial roles in homeostasis and disease. Accurate recapitulation of the chemical, structural, and mechanical properties of the ECM in brain cell cultures is essential for the development of translatable in vitro models; however, thus far, this task has proven highly challenging. This review provides an in‐depth discussion of this challenge, including an overview of the properties of the ECM that in vitro models should endeavour to capture, a survey of ECM analogues that are currently used for this purpose, and a discussion of the main hindrances to developing more effective ECM‐like coating materials.

Protein pathway analysis to study development-dependent effects of acute and repeated trimethyltin (TMT) treatments in 3D rat brain cell cultures

Trimethyltin is an organometallic compound, described to be neurotoxic and to trigger neuroinflammation and oxidative stress. Previous studies associated TMT with the perturbation of mitochondrial function, or neurotransmission. However, the mechanisms of toxicity may differ depending on the duration of exposure and on the stage of maturation of brain cells. This study aim at elucidating whether the toxicity pathways triggered by a known neurotoxicant (TMT) differs depending on cell maturation stage or duration of exposure. To this end omics profiling of immature and differentiated 3D rat brain cell cultures exposed for 24 h or 10 days (10-d) to 0.5 and 1 μM of TMT was performed to better understand the underlying mechanisms of TMT associated toxicity.Proteomics identified 55 and 17 proteins affected by acute TMT treatment in immature and differentiated cultures respectively, while 10-day treatment altered 96 proteins in immature cultures versus 353 in differentiated. The results suggest different sensitivity to TMT depending on treatment duration and cell maturation. In accordance with known TMT mechanisms oxidative stress and neuroinflammation was observed after 10-d treatment at both maturation stages, whereas the neuroinflammatory process was more prominent in differentiated cultures than in the immature, no development-dependent difference could be detected for oxidative stress or synaptic neurodegeneration. Pathway analysis revealed that both vesicular trafficking and the synaptic machinery were strongly affected by 10-d TMT treatment in both maturation stages, as was GABAergic and glutamatergic neurotransmission.This study shows that omics approaches combined with pathway analysis constitutes an improved tool-set in elucidating toxicity mechanisms.

Combination of Genetics and Nanotechnology for Down Syndrome Modification: A Potential Hypothesis and Review of the Literature

Down syndrome (DS) is one of the most prevalent genetic disorders in humans. The use of new approaches in genetic engineering and nanotechnology methods in combination with natural cellular phenomenon can modify the disease in affected people. We consider two CRISPR/Cas9 systems to cut a specific region from short arm of the chromosome 21 (Chr21) and replace it with a novel designed DNA construct, containing the essential genes in chromatin remodeling for inactivating of an extra Chr21. This requires mimicking of the natural cellular pattern for inactivation of the extra X chromosome in females. By means of controlled dosage of an appropriate Nano-carrier (a surface engineered Poly D, L-lactide-co-glycolide (PLGA) for integrating the relevant construct in Trisomy21 brain cell culture media and then in DS mouse model, we would be able to evaluate the modification and the reduction of the active extra Chr21 and in turn reduce substantial adverse effects of the disease, like intellectual disabilities. The hypothesis and study seek new insights in Down syndrome modification.

Hydrogenized Water Effects on Protection of Brain Cells from Oxidative Stress and Glutamate Toxicity

Hydrogenized water is known to have protective effects on cells and tissues, mainly through its antioxidant activities. Here we examined the protective effects of a commercial source of hydrogenized water on cultured human brain cells. Hydrogenized water was able to protect brain cells from oxidative stress and glutamate toxicity. At H2 concentrations above 0.01 mM the glutathione levels increased in cultured brain cells. The level of glutathione rose from approximately 500 to approximately 850 μM at the maximum dose of hydrogenized water with an EC50 of approximately 0.030 mM. Hydrogenized water was also able to enhance the signaling pathway for oxidative stress response mediated by Nrf2 (Nuclear factor erythroid 2 like factor). Treatment of cells with hydrogenized water at concentrations above 0.01 mM H2 induced activation of Nrf2 (EC50 approximately 0.05 mM). Hydrogenized water was also able to protect brain cells against glutamate toxicity. Using a DNA damage response element, (γH2AX, to monitor the damage of glutamate toxicity we found that concentrations of H2 above 0.01 mM protected cells from glutamate damage with an EC50 of approximately 0.05 mM H2. These in vitro results demonstrated that hydrogenized water can protect brain cells against common types of damage from oxidative stress and glutamate toxicity.

In vitro models to study insulin and glucocorticoids modulation of trimethyltin (TMT)-induced neuroinflammation and neurodegeneration, and in vivo validation in db/db mice.

Brain susceptibility to a neurotoxic insult may be increased in a compromised health status, such as metabolic syndrome. Both metabolic syndrome and exposure to trimethyltin (TMT) are known to promote neurodegeneration. In combination the two factors may elicit additive or compensatory/regulatory mechanisms. Combined effects of TMT exposure (0.5-1μM) and mimicked metabolic syndrome-through modulation of insulin and glucocorticoid (GC) levels-were investigated in three models: tridimensional rat brain cell cultures for neuron-glia effects; murine microglial cell line BV-2 for a mechanistic analysis of microglial reactivity; and db/db mice as an in vivo model of metabolic syndrome. In 3D cultures, low insulin condition significantly exacerbated TMT's effect on GABAergic neurons and promoted TMT-induced neuroinflammation, with increased expression of cytokines and of the regulator of intracellular GC activity, 11β-hydroxysteroid dehydrogenase 1 (11β-Hsd1). Microglial reactivity increased upon TMT exposure in medium combining low insulin and high GC. These results were corroborated in BV-2 microglial cells where lack of insulin exacerbated the TMT-induced increase in 11β-Hsd1 expression. Furthermore, TMT-induced microglial reactivity seems to depend on mineralocorticoid receptor activation. In diabetic BKS db mice, a discrete exacerbation of TMT neurotoxic effects on GABAergic neurons was observed, together with an increase of interleukin-6 (IL-6) and of basal 11β-Hsd1 expression as compared to controls. These results suggest only minor additive effects of the two brain insults, neurotoxicant TMT exposure and metabolic syndrome conditions, where 11β-Hsd1 appears to play a key role in the regulation of neuroinflammation and of its protective or neurodegenerative consequences.

SUN-098 Goldfish Insulin: Molecular Cloning, Hepatic Regulation by Spexin, and Mechanisms for Feeding Control in Fish Model

Insulin is a key hormone for metabolism and glucose homeostasis. In mammals, it also acts as a satiety factor with inhibitory effect on food intake, but its biological actions in appetite control in lower vertebrates is still unknown. Spexin (SPX), a neuropeptide with pleotropic functions, has been recently confirmed to be a novel satiety factor in fish model via functional coupling with insulin, although the details of their interactions have yet to be elucidated. In this study, using goldfish as a model, the mechanism for feeding regulation by insulin and its functional interactions with SPX at hepatic level were examined. As a first step, goldfish insulin was cloned and found to be widely expressed at tissue level, especially with high levels of expression in the liver and visceral fat. In silico modeling also showed that the 3D structure of the mature peptide of goldfish insulin was highly comparable if not identical to its human counterpart. In goldfish liver cell culture, insulin mRNA level could be up-regulated by SPX treatment via PLC/IP3/PKC and Ca2+/CaM/CaMK-II pathways, while the opposite was true by removing endogenous SPX using immunoneutralization with SPX antiserum. In whole animal experiment, IP injection with insulin was found to inhibit feeding behavior and food consumption in goldfish. Similar treatment in vivo was also effective in elevating POMC, CART, CCK and leptin mRNA levels with concurrent drop in NPY, AGRP and apelin transcript expression in the telencephalon, optic tectum and hypothalamus, the brain areas known to be involved in feeding control in fish models. Using brain cell culture prepared from these three areas, similar changes in transcript expression for the respective orexigenic and anorexigenic factors were also noted with insulin treatment in a time- and dose-dependent manner. These findings, as a whole, suggest that SPX can act in an autocrine/paracrine manner to induce insulin expression in goldfish liver and the subsequence rise of insulin in circulation presumably can inhibit food intake through differential regulation of orexigenic/anorexigenic signals expressed in brain areas involved in appetite control in fish model.

Argininosuccinate neurotoxicity and prevention by creatine in argininosuccinate lyase deficiency: An in vitro study in rat three-dimensional organotypic brain cell cultures.

The urea cycle disorder (UCD) argininosuccinate lyase (ASL) deficiency, caused by a defective ASL enzyme, exhibits a wide range of phenotypes, from life-threatening neonatal hyperammonemia to asymptomatic patients, with only the biochemical marker argininosuccinic acid (ASA) elevated in body fluids. Remarkably, even without ever suffering from hyperammonemia, patients often develop severe cognitive impairment and seizures. The goal of this study was to understand the effect on the known toxic metabolite ASA and the assumed toxic metabolite guanidinosuccinic acid (GSA) on developing brain cells, and to evaluate the potential role of creatine (Cr) supplementation, as it was described protective for brain cells exposed to ammonia. We used an in vitro model, in which we exposed three-dimensional (3D) organotypic rat brain cell cultures in aggregates to different combinations of the metabolites of interest at two time points (representing two different developmental stages). After harvest and cryopreservation of the cell cultures, the samples were analyzed mainly by metabolite analysis, immunohistochemistry, and western blotting. ASA and GSA were found toxic for astrocytes and neurons. This toxicity could be reverted in vitro by Cr. As well, an antiapoptotic effect of ASA was revealed, which could contribute to the neurotoxicity in ASL deficiency. Further studies in human ASL deficiency will be required to understand the biochemical situation in the brain of affected patients, and to investigate the impact of high or low arginine doses on brain Cr availability. In addition, clinical trials to evaluate the beneficial effect of Cr supplementation in ASL deficiency would be valuable.

Kultur Sel Otak Ikan Kerapu Bebek (Cromileptes altivelis) Dengan Menggunakan Media L-15, MEM Dan TCM <br><i>[Cell Culture Brain Of Chromileptes altivelis With Medium L-15, MEM And TCM]<i>.

Abstract Humpback grouper is high value commodity fish. The demand of domestic and foreign markets increases every year. The obstacles of Humpback grouper fish cultivation is a disease caused by Viral Nervous Necrosis (VNN). The virus attacks the brain leading to mass death. To prevent the disease, a research on VNN virus has to be started. The problem of the research is the lack of culture of fish tissue. Virus have specific location to tissue cell and species to be infected. Thereofore, first step must be done is growth from tissue cell which infection of virus. Cell culture is a technique to isolate cell, protoplasm, tissue or organ to be grown in a sterile condition with nutrition containing growth hormone to promote multiplication. Cell culture technique can be used in many basic applications in cell biology. The purpose of this research is to develop cell culture from Grouper fish brain for VNN research. The hope is culture can grow and develop well.Research executed at June until Desember 2009 in Laboratory In Vitro of Veterinari Faculty of Airlangga University and Laboratory Gastroenteritis Institute of Tropical Disease, Airlangga University, Surabaya. The result showed that brain cell culture of humpback grouper fish can be grow and multiply well in all three media, media L-15 cell growth was faster compared to media MEM and TCM.

Ammonium accumulation and chemokine decrease in culture media of Gcdh−/− 3D reaggregated brain cell cultures

Glutaric Aciduria type I (GA-I) is caused by mutations in the GCDH gene. Its deficiency results in accumulation of the key metabolites glutaric acid (GA) and 3-hydroxyglutaric acid (3-OHGA) in body tissues and fluids. Present knowledge on the neuropathogenesis of GA-I suggests that GA and 3-OHGA have toxic properties on the developing brain.We analyzed morphological and biochemical features of 3D brain cell aggregates issued from Gcdh−/− mice at two different developmental stages, day-in-vitro (DIV) 8 and 14, corresponding to the neonatal period and early childhood. We also induced a metabolic stress by exposing the aggregates to 10 mM l-lysine (Lys).Significant amounts of GA and 3-OHGA were detected in Gcdh−/− aggregates and their culture media. Ammonium was significantly increased in culture media of Gcdh−/− aggregates at the early developmental stage. Concentrations of GA, 3-OHGA and ammonium increased significantly after exposure to Lys. Gcdh−/− aggregates manifested morphological alterations of all brain cell types at DIV 8 while at DIV 14 they were only visible after exposure to Lys. Several chemokine levels were significantly decreased in culture media of Gcdh−/− aggregates at DIV 14 and after exposure to Lys at DIV 8.This new in vitro model for brain damage in GA-I mimics well in vivo conditions. As seen previously in WT aggregates exposed to 3-OHGA, we confirmed a significant ammonium production by immature Gcdh−/− brain cells. We described for the first time a decrease of chemokines in Gcdh−/− culture media which might contribute to brain cell injury in GA-I.

Quantification of Alpha-Synuclein in Biological Fluids by Electrochemiluminescence-Based Detection.

Several potential marker candidates for Parkinson's disease (PD) in cerebrospinal fluid (CSF) have been identified. These include α-synuclein, a major constituent of the intracellular aggregates Lewy bodies, a neuropathological hallmark of PD, and others. The extracellular presence of α-synuclein in blood, CSF, saliva, and conditioned media motivated the hypothesis that the quantification of CSF α-synuclein is a biomarker for α-synuclein-related disorders. We here describe the development of an electrochemiluminescence-based assay by conversion of an established ELISA for quantification of α-synuclein. The assay not only works with CSF but can also be used to quantify α-synuclein in different biological fluids, i.e., whole blood and blood products, saliva, as well as brain homogenates and cell culture material.

295. A 3T MRS in the analysis of cell differentiation induced in human Neuroblastoma cells treated with l-Acetylaspartate and electro stimulation

Purpose A 1H-MRS spectrum on a patient’s healthy brain differs on the pathological part of the same organ. L-Acetylaspartate (NAA), due to its high concentration in the brain, returns intense MRS spectrum signal, reducing its intensity more or less drastically in patients suffering from various brain diseases. Even cultures of brain cells have the same spectrum as man. Because brain cells function with specific electrical impulses, we electrically stimulated the brain cells of a tumour, in the presence of NAA, to attempt to modify their MRS spectrum from pathological to normal. Methods Hodgkin and Huxley (1963) have experimentally documented on the cellular membrane a ddp (ΔV) of −90 mV. The whole human organism starting from the cells to organs and apparatus behaves like a more or less complex system of RLC circuits. The T.H. Bullock (1959) defines three different types of electrical potentials: pacemaker; transduction; action. When electrical impulse crossing the cell’s RC circuit are modified if the cell has low resistance ( norm-functional) or high resistance (pathology). These have suggested the idea to compensate, in the pathological tissue, a well-defined molar doses of the NAA. TENS has shown, on humans, that the square shape of the signal is physiologically the most advantageous for several reasons. The research was carried out on human Neuroblastoma cells, provided by the University Hospital of MANASSAS (Virginia) for research purposes only. The human cell cultures of Neuroblastoma were placed on multiwall plates with a cell culture medium substrate. The procedure, on three sets of plates with the same characteristics, is divided into three phases: • 1st PHASE: suitable molar concentration of L-Acetylaspartate (NAA); • 2nd PHASE: cell plates treated with NAA, concentration of 4 mM, untreated cell plates (controls) received electro stimulation with different times (20, 40, 60 min); • 3rd PHASE: An equal number of plates, treated and not with NAA, were not electro stimulated. The plates had cells, cells treated with NAA, culture medium, culture medium and NAA. All samples underwent nuclear magnetic resonance spectroscopy. Preliminary results The spectra, after the different electro stimulations, were acquired at 24 and 48 h with a human 3T MRS scanner. see Fig. 1 Conclusions the spectra of pathological cells treated with NAA and electro stimulation for 60’, revealed to be of normal appearance.

Resistance of pearlspot larvae, Etroplus suratensis, to redspotted grouper nervous necrosis virus by immersion challenge.

Viral nervous necrosis (VNN) affects more than 120 species mostly belonging to the order Perciformes. However, none of the brackishwater species belonging to the family Cichlidae under the order Perciformes are reported to be susceptible. Hence, the present experiment was undertaken to study the susceptibility of the brackishwater cichlid, pearlspot, Etroplus suratensis to NNV. Thirty-day-old pearlspot larvae were infected with NNV by immersion. Mortality was recorded till 14days post-infection, and the infected larvae were subjected to nested RT-PCR and histology. The virus was isolated from infected larvae using SSN-1 cells. To study the replication of the virus in vitro, primary cultured brain cells of E.suratensis and IEK cells were infected with NNV. No mortality was observed in any of the control or experimentally infected larvae. However, the experimentally infected larvae were positive for NNV by nested RT-PCR and the virus was isolated using SSN-1 cells. Further, the infected pearlspot brain cells and IEK cells showed cytopathic effect at second and third passage of the virus and they were positive for NNV by nested RT-PCR. Pearlspot is relatively resistant to VNN although the virus could replicate in the larvae and in cell culture.

Neuronal Cells Confinement by Micropatterned Cluster-Assembled Dots with Mechanotransductive Nanotopography.

Artificially grown neuronal cultures of brain cells have been used for decades in the attempt to reproduce and study in vitro the complexity of brain circuits. It soon became evident that this alone was insufficient, because of the random architecture of these artificial networks. Important groundwork therefore resulted in the development of methods to confine neuronal adhesion at specific locations to match predefined network topologies and connectivity. Despite this notable progress in neural circuitry engineering, there is still need for micropatterned substrates that recapitulate better biophysical cues of the neuronal microenvironment, taking into account recent findings of their significance for neuronal differentiation and functioning. Here, we report the development and characterization of a novel approach that, by using supersonic cluster beam deposition of zirconia nanoparticles, allows the patterning of small nanostructured cell-adhesive areas according to predefined geometries onto elsewhere nonadhesive antifouling glass surfaces. As distinguishing features, compared to other micropatterning approaches in this context, the integrated nanostructured surfaces possess extracellular matrix-like nanotopographies of predetermined roughness; previously shown to be able to promote neuronal differentiation due to their impact on mechanotransductive processes, and can be used in their original state without any coating requirements. These micropatterned substrates were validated using (i) a neuron-like PC12 cell line and (ii) primary cultures of rat hippocampal neurons. After initial uniform plating, both neuronal cells types were found to converge and adhere specifically to the nanostructured regions. The cell-adhesive areas effectively confined cells, even when these were highly mobile and repeatedly attempted to cross boundaries. Inside these small permissive islands, cells grew and differentiated, in the case of the hippocampal neurons, up to the formation of mature, functionally active, and highly connected synaptic networks. In addition, when spontaneous instances of axon bridging between nearby dots occurred, a functional interdot communication between these subgroups of cells was observed.

Longitudinal investigation of the metabolome of 3D aggregating brain cell cultures at different maturation stages by 1H HR-MAS NMR

The aim of the present study was to establish the developmental profile of metabolic changes of 3D aggregating brain cell cultures by 1H high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. The histotypic 3D brain aggregate, containing all brain cell types, is an excellent model for mechanistic studies including OMICS analysis; however, their metabolic profile has not been yet fully investigated. Chemometric analysis revealed a clear separation of samples from the different maturation time points. Metabolite concentration evolutions could be followed and revealed strong and various metabolic alterations. The strong metabolite evolution emphasizes the brain modeling complexity during maturation, possibly reflecting physiological processes of brain tissue development. The small observed intra- and inter-experimental variabilities show the robustness of the combination of 1H-HR-MAS NMR and 3D brain aggregates, making it useful to investigate mechanisms of toxicity that will ultimately contribute to improve predictive neurotoxicology. Graphical Abstract ᅟ.

How to Study the Uptake and Toxicity of Nanoparticles in Cultured Brain Cells: The Dos and Don't Forgets.

Due to their exciting properties, engineered nanoparticles have obtained substantial attention over the last two decades. As many types of nanoparticles are already used for technical and biomedical applications, the chances that cells in the brain will encounter nanoparticles have strongly increased. To test for potential consequences of an exposure of brain cells to engineered nanoparticles, cell culture models for different types of neural cells are frequently used. In this review article we will discuss experimental strategies and important controls that should be used to investigate the physicochemical properties of nanoparticles for the cell incubation conditions applied as well as for studies on the biocompatibility and the cellular uptake of nanoparticles in neural cells. The main focus of this article will be the interaction of cultured neural cells with iron oxide nanoparticles, but similar considerations are important for studying the consequences of an exposure of other types of cultured cells with other types of nanoparticles. Our article aims to improve the understanding of the special technical challenges of working with nanoparticles on cultured neural cells, to identify potential artifacts and to prevent misinterpretation of data on the potential adverse or beneficial consequences of a treatment of cultured cells with nanoparticles.