Primary Neuronal Cell Cultures Research Articles

Detrimental Effects of HMGB-1 Require Microglial-Astroglial Interaction: Implications for the Status Epilepticus -Induced Neuroinflammation.

Temporal Lobe Epilepsy (TLE) is the most common form of human epilepsy and available treatments with antiepileptic drugs are not disease-modifying therapies. The neuroinflammation, neuronal death and exacerbated plasticity that occur during the silent period, following the initial precipitating event (IPE), seem to be crucial for epileptogenesis. Damage Associated Molecular Patterns (DAMP) such as HMGB-1, are released early during this period concomitantly with a phenomenon of reactive gliosis and neurodegeneration. Here, using a combination of primary neuronal and glial cell cultures, we show that exposure to HMGB-1 induces dendrite loss and neurodegeneration in a glial-dependent manner. In glial cells, loss of function studies showed that HMGB-1 exposure induces NF-κB activation by engaging a signaling pathway that involves TLR2, TLR4, and RAGE. In the absence of glial cells, HMGB-1 failed to induce neurodegeneration of primary cultured cortical neurons. Moreover, purified astrocytes were unable to fully respond to HMGB-1 with NF-κB activation and required microglial cooperation. In agreement, in vivo HMGB-1 blockage with glycyrrhizin, immediately after pilocarpine-induced status epilepticus (SE), reduced neuronal degeneration, reactive astrogliosis and microgliosis in the long term. We conclude that microglial-astroglial cooperation is required for astrocytes to respond to HMGB-1 and to induce neurodegeneration. Disruption of this HMGB-1 mediated signaling pathway shows beneficial effects by reducing neuroinflammation and neurodegeneration after SE. Thus, early treatment strategies during the latency period aimed at blocking downstream signaling pathways activated by HMGB-1 are likely to have a significant effect in the neuroinflammation and neurodegeneration that are proposed as key factors in epileptogenesis.

Polyamide 6.6 thin films with distinct ratios of the main chemical groups: Influence in the primary neuronal cell culture

Neuronal cell cultures have been instrumental to the advance of our knowledge in a wide range of areas. One of the limitations of this model is the lack of stability, over time, of the substrate materials that are used in neuronal cell cultures. In this work, sputtering was used to permanently modify glass coverslips with a polyamide 6.6 thin film, using different discharge gases. Differences in the chemical composition of the films, resulting from the conditions used in deposition, were correlated with the surface properties. After characterization by SEM, TEM, AFM, XPS, zeta-potential, wettability and evaluation of the mechanical properties, some surfaces were selected for in vitro testing in the development of cultured cerebrocortical neurons. The results showed that the chemical composition and surface charge are particularly affected by the variation of the deposition parameters, although wettability, for example, did not present significant variations. In vitro tests revealed that the surfaces with higher NH 2 /COOH ratio allowed the adhesion and differentiation of neurons, with a morphology similar to that obtained after plating of the cells on poly- d -lysine-coated glass. This work opens new perspectives for the permanent modification of materials towards multifunctional neural compatible surfaces and open new areas for bioengineering applications. • Polyamide 6.6 thin films for permanent modification of glass surface for neurons adhesion • The surface of the deposited material can be preconditioned during deposition. • The tuning of surface properties potentially ensures more stable materials for cell attachment. • Polyamide permanent modified glass present similar results to the standard PDL-glass.

Proteasomal Inhibition Redirects the PrP-Like Shadoo Protein to the Nucleus

The Shadoo protein (Sho) exhibits homology to the hydrophobic region of the cellular isoform of prion protein (PrPC). As prion-infected brains gradually accumulate infectivity-associated isoforms of prion protein (PrPSc), levels of mature endogenous Sho become reduced. To study the regulatory effect of the proteostatic network on Sho expression, we investigated the action of lactacystin, MG132, NH4Cl, and 3-methyladenine (3-MA) in two cell culture models. In primary mixed neuronal and glial cell cultures (MNGCs) from transgenic mice expressing wild-type Sho from the PrP gene promoter (Tg.Sprn mice), lactacystin- and MG132-mediated inhibition of proteasomal activity shifted the repertoire of Sho species towards unglycosylated forms appearing in the nuclei; conversely, the autophagic modulators NH4Cl and 3-MA did not affect Sho or PrPC glycosylation patterns. Mouse N2a neuroblastoma cells expressing Sho under control of a housekeeping gene promoter treated with MG132 or lactacystin also showed increased nuclear localization of unglycosylated Sho. As two proteasomal inhibitors tested in two cell paradigms caused redirection of Sho to nuclei at the expense of processing through the secretory pathway, our findings define a balanced shift in subcellular localization that thereby differs from the decreases in net Sho species seen in prion-infected brains. Our data are indicative of a physiological pathway to access Sho functions in the nucleus under conditions of impaired proteasomal activity. We also infer that these conditions would comprise a context wherein Sho’s N-terminal nucleic acid–binding RGG repeat region is brought into play.

Optically Induced Calcium-Dependent Gene Activation and Labeling of Active Neurons Using CaMPARI and Cal-Light.

The advent of optogenetic methods has made it possible to use endogeneously produced molecules to image and manipulate cellular, subcellular, and synaptic activity. It has also led to the development of photoactivatable calcium-dependent indicators that mark active synapses, neurons, and circuits. Furthermore, calcium-dependent photoactivation can be used to trigger gene expression in active neurons. Here we describe two sets of protocols, one using CaMPARI and a second one using Cal-Light. CaMPARI, a calcium-modulated photoactivatable ratiometric integrator, enables rapid network-wide, tunable, all-optical functional circuit mapping. Cal-Light, a photoactivatable calcium sensor, while slower to respond than CaMPARI, has the capacity to trigger the expression of genes, including effectors, activators, indicators, or other constructs. Here we describe the rationale and provide procedures for using these two calcium-dependent constructs (1) in vitro in dissociated primary neuronal cell cultures (CaMPARI & Cal-Light); (2) in vitro in acute brain slices for circuit mapping (CaMPARI); (3) in vivo for triggering photoconversion or gene expression (CaMPARI & Cal-Light); and finally, (4) for recovering photoconverted neurons post-fixation with immunocytochemistry (CaMPARI). The approaches and protocols we describe are examples of the potential uses of both CaMPARI & Cal-Light. The ability to mark and manipulate neurons that are active during specific epochs of behavior has a vast unexplored experimental potential.

Bioactive molecules isolated from olive pomace extract protect murine cortex neurons from NMDA‐mediated cell death

Here we demonstrate that olive pomace extracts contain bioactive molecules able to prevent primary neuron death induced by altered calcium homeostasis.We have observed previously that partially purified extracts counteracted calcium induced cell damages on human neuroblastoma (SK‐N‐BE) and mouse brain endothelioma (bEnd5) cell lines operating through the regulation of the cell dynamics that involve the calcium ions.It is well known that changes in [Ca2+]i are crucial events during signal transduction processes involved in several cellular physiological functions. Moreover, even a limited dysregulation in intracellular Ca2+ homeostasis is related to several acute and chronic diseases including neurodegenerations. In this context, we have now tested the effect of the bioactive molecules, detected in olive pomace, on primary cell cultures of murine cortex neurons, used as a cellular model. Accordingly, in order to induce a cytotoxic influx of Ca2+ through opening of the NMDA receptor, we treated neurons s with NMDA (N‐Methyl‐D‐Aspartic‐Acid) for 24 hour. When this cell treatment was carried out in the presence of preparations of the bioactive molecules, the effect of NMDA, evaluated as percentage of cell death, was significantly reduced. Moreover, following cell exposure to NMDA in the absence or the presence of the bioactive molecules, the [Ca2+]i was measured using different alternative techniques. The results obtained confirmed that these bioactive preparations act by molecular mechanisms that prevent the alteration of the intracellular calcium homeostasis mediated by NMDA. In conclusion, these bioactive molecules protect murine cortex neurons by reducing the toxic effects of [Ca2+]i, operated by the NMDA receptor. Further investigations are in progress to understand if the bioactive olive pomace derived molecules act directly on this receptor or on pathways activated downstream its opening. At present, the issue we give the highest priority is to identify the molecular nature of the relevant bioactive compounds. To solve this question we are carrying out mass spectrometry analyses on differently purified extracts.The ultimate aim is to achieve definitive conclusions about the potential practical applications for these natural molecules in their purified form. Indeed, providing comprehensive information about the structural and functional aspects of these molecules is crucial to propose new possible strategies for therapeutic interventions in neurodegenerations.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Stimulation of noradrenergic transmission by reboxetine is beneficial for a mouse model of progressive parkinsonism

Parkinson’s disease (PD) is the second most common neurodegenerative disorder and is characterized by motor deficits such as tremor, rigidity and bradykinesia. These symptoms are directly caused by the loss of dopaminergic neurons. However, a wealth of clinical evidence indicates that the dopaminergic system is not the only system affected in PD. Postmortem studies of brains from PD patients have revealed the degeneration of noradrenergic neurons in the locus coeruleus (LC) to the same or even greater extent than that observed in the dopaminergic neurons of substantia nigra (SN) and ventral tegmental area (VTA). Moreover, studies performed on rodent models suggest that enhancement of noradrenergic transmission may attenuate the PD-like phenotype induced by MPTP administration, a neurotoxin-based PD model. The aim of this study was to investigate whether chronic treatment with either of two compounds targeting the noradrenergic system (reboxetine or atipamezole) possess the ability to reduce the progression of a PD-like phenotype in a novel mouse model of progressive dopaminergic neurodegeneration induced by the genetic inhibition of rRNA synthesis in dopaminergic neurons, mimicking a PD-like phenotype. The results showed that reboxetine improved the parkinsonian phenotype associated with delayed progression of SN/VTA dopaminergic neurodegeneration and higher dopamine content in the striatum. Moreover, the alpha1-adrenergic agonist phenylephrine enhanced survival of TH+ neurons in primary cell cultures, supporting the putative neuroprotective effects of noradrenergic stimulation. Our results provide new insights regarding the possible influence of the noradrenergic system on dopaminergic neuron survival and strongly support the hypothesis regarding the neuroprotective role of noradrenaline.

Posterior ventral tegmental area-nucleus accumbens shell circuitry modulates response to novelty.

Dopamine release in the nucleus accumbens from ventral tegmental area (VTA) efferent neurons is critical for orientation and response to novel stimuli in the environment. However, there are considerable differences between neuronal populations of the VTA and it is unclear which specific cell populations modulate behavioral responses to environmental novelty. A retroDREADDs (designer drugs exclusively activated by designer receptors) technique, comprising designer G protein-coupled receptors exclusively activated by designer drugs and modulated by retrograde transported Cre, was used to selectively stimulate neurons of the VTA which project to the nucleus accumbens shell (AcbSh). First, the selectivity and expression of the human M3 muscarinic receptor-based adeno-associated virus (AAV-hM3D) was confirmed in primary neuronal cell cultures. Second, AAV-CMV-GFP/Cre was infused into the AcbSh and AAV-hSyn-DIO-hM3D(Gq)-mCherry (a presynaptic enhancer in the presence of its cognate ligand clozapine-N-oxide) was infused into the VTA of ovariectomized female Fisher 344 rats to elicit hM3D(Gq)-mCherry production specifically in neurons of the VTA which synapse in the AcbSh. Finally, administration of clozapine-N-oxide significantly altered rodents’ response to novelty (e.g. absence of white background noise) by activation of hM3D(Gq) receptors, without altering gross locomotor activity or auditory processing per se. Confocal imaging confirmed production of mCherry in neurons of the posterior aspect of the VTA (pVTA) suggesting these neurons contribute to novelty responses. These results suggest the pVTA-AcbSh circuit is potentially altered in motivational disorders such as apathy, depression, and drug addiction. Targeting the pVTA-AcbSh circuit, therefore, may be an effective target for pharmacological management of such psychopathologies.

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.

Effect of Silk Fibroin on Neuroregeneration After Traumatic Brain Injury.

Traumatic brain injury is one of the leading causes of disability among the working-age population worldwide. Despite attempts to develop neuroprotective therapeutic approaches, including pharmacological or cellular technologies, significant advances in brain regeneration have not yet been achieved. Development of silk fibroin-based biomaterials represents a new frontier in neuroregenerative therapies after brain injury. In this study, we estimated the short and long-term effects of silk fibroin scaffold transplantation on traumatic brain injury and biocompatibility of this biomaterial within rat neuro-vascular cells. Silk fibroin microparticles were injected into a brain damage area 1day after the injury. Silk fibroin affords neuroprotection as judged by diminished brain damage and recovery of long-term neurological functions. We did not detect considerable toxicity to neuro-vascular cells cultured on fibroin/fibroin-gelatin microparticles in vitro. Cultivation of primary cell cultures of neurons and astrocytes on silk fibroin matrices demonstrated their higher viability under oxygen-glucose deprivation compared to 2D conditions on plastic plates. Thus, we conclude that scaffolds based on silk fibroin can become the basis for the creation of constructs aimed to treat brain regeneration after injury.

Peptide-siRNA Supramolecular Particles for Neural Cell Transfection.

Small interfering ribonucleic acid (siRNA)‐based gene knockdown is an effective tool for gene screening and therapeutics. However, the use of nonviral methods has remained an enormous challenge in neural cells. A strategy is reported to design artificial noncationic modular peptides with amplified affinity for siRNA via supramolecular assembly that shows efficient protein knockdown in neural cells. By solid phase synthesis, a sequence that binds specifically double‐stranded ribonucleic acid (dsRNA) with a self‐assembling peptide for particle formation is integrated. These supramolecular particles can be further functionalized with bioactive sequences without affecting their biophysical properties. The peptide carrier is found to silence efficiently up to 83% of protein expression in primary astroglial and neuronal cell cultures without cytotoxicity. In the case of neurons, a reduction in electrical activity is observed once the presynaptic protein synaptophysin is downregulated by the siRNA–peptide particles. The results demonstrate that the supramolecular particles offer an siRNA delivery platform for efficient nonviral gene screening and discovery of novel therapies for neural cells.

Differential effects of hypothermia on neurovascular unit determine protective or toxic results: Toward optimized therapeutic hypothermia

Therapeutic hypothermia (TH) benefits survivors of cardiac arrest and neonatal hypoxic–ischemic injury and may benefit stroke patients. Large TH clinical trials, however, have shown mixed results. Given the substantial pre-clinical literature supporting TH, we explored possible mechanisms for clinical trial variability. Using a standard rodent stroke model (n = 20 per group), we found smaller infarctions after 2 h pre- or post-reperfusion TH compared to 4 h. To explore the mechanism of this discrepancy, we used primary cell cultures of rodent neurons, astrocytes, or endothelial cells subjected to oxygen–glucose deprivation (OGD). Then, cells were randomly assigned to 33℃, 35℃ or 37℃ for varying durations after varying delay times. Both 33 and 35℃ TH effectively preserved all cell types, although 33℃ was superior. Longer cooling durations overcame moderate delays to cooling initiation. In contrast, TH interfered with astrocyte paracrine protection of neurons in a temperature-dependent manner. These findings suggest that longer TH is needed to overcome delays to TH onset, but shorter TH durations may be superior to longer, perhaps due to suppression of astrocytic paracrine support of neurons during injury. We propose a scheme for optimizing TH after cerebral injury to stimulate further studies of cardiac arrest and stroke.

Contemporary Circulating Enterovirus D68 Strains Have Acquired the Capacity for Viral Entry and Replication in Human Neuronal Cells.

Enterovirus D68 (EV-D68) has historically been associated with respiratory illnesses. However, in the summers of 2014 and 2016, EV-D68 outbreaks coincided with a spike in polio-like acute flaccid myelitis/paralysis (AFM/AFP) cases. This raised concerns that EV-D68 could be the causative agent of AFM during these recent outbreaks. To assess the potential neurotropism of EV-D68, we utilized the neuroblastoma-derived neuronal cell line SH-SY5Y as a cell culture model to determine if differential infection is observed for different EV-D68 strains. In contrast to HeLa and A549 cells, which support viral infection of all EV-D68 strains tested, SH-SY5Y cells only supported infection by a subset of contemporary EV-D68 strains, including isolates from the 2014 outbreak. Viral replication and infectivity in SH-SY5Y were assessed using multiple assays: virus production, cytopathic effects, cellular ATP release, and VP1 capsid protein production. Similar differential neurotropism was also observed in differentiated SH-SY5Y cells, primary human neuron cultures, and a mouse paralysis model. Using the SH-SY5Y cell culture model, we determined that barriers to viral binding and entry were at least partly responsible for the differential infectivity phenotype. Transfection of genomic RNA into SH-SY5Y generated virions for all EV-D68 isolates, but only a single round of replication was observed from strains that could not directly infect SH-SY5Y. In addition to supporting virus replication and other functional studies, this cell culture model may help identify the signatures of virulence to confirm epidemiological associations between EV-D68 strains and AFM and allow for the rapid identification and characterization of emerging neurotropic strains.IMPORTANCE Since the EV-D68 outbreak during the summer of 2014, evidence of a causal link to a type of limb paralysis (AFM) has been mounting. In this article, we describe a neuronal cell culture model (SH-SY5Y cells) in which a subset of contemporary 2014 outbreak strains of EV-D68 show infectivity in neuronal cells, or neurotropism. We confirmed the difference in neurotropism in vitro using primary human neuron cell cultures and in vivo with a mouse paralysis model. Using the SH-SY5Y cell model, we determined that a barrier to viral entry is at least partly responsible for neurotropism. SH-SY5Y cells may be useful in determining if specific EV-D68 genetic determinants are associated with neuropathogenesis, and replication in this cell line could be used as rapid screening tool for identification of neurotropic EV-D68 strains. This may assist with better understanding of pathogenesis and epidemiology and with the development of potential therapies.

Irisin Exerts Neuroprotective Effects on Cultured Neurons by Regulating Astrocytes

Neurons suffer detrimental effects from β-amyloid toxicity in Alzheimer's disease. The exercise hormone, irisin, is found to induce a neuroprotective gene program and facilitates the beneficial effects on cognitive function. But no effort is made to test its direct protective effects on neurons against the Aβ-induced cell toxicity so far. In the present study, we investigated whether irisin could protect neurons against Aβ- (25–35) induced cell damage and explored the possible underlying mechanisms. Primary cell cultures of astrocytes and neurons were established. Conditioned medium from astrocyte was collected for the treatment and biochemistry assay study. To explore the protein expression changes, Western blot and ELISA assays were used in these in vitro cell culture models. Exposure of hippocampal neurons to 10 μM Aβ (25–35) caused significant reduction on cell viability, and the toxic effect was not significantly reduced by the coadministration of irisin. However, pretreated astrocyte-conditioned medium with irisin for 12 hours notably protected the neurons from the toxicity of Aβ. Also, we found that irisin could attenuate the release of IL-6 and IL-1β from cultured astrocytes and decrease the expression level of COX-2 and phosphorylation of AKT. Last, we found that irisin could reduce NFκB activation in astrocyte exposed to Aβ by preventing the phosphorylation and the loss of IκBα. Our finding may provide novel evidence for the future application of irisin in the treatment of Alzheimer's disease and the memory dysfunction in diabetes mellitus.

Perinatal Asphyxia and Brain Development: Mitochondrial Damage Without Anatomical or Cellular Losses

Perinatal asphyxia remains a significant cause of neonatal mortality and is associated with long-term neurodegenerative disorders. In the present study, we evaluated cellular and subcellular damages to brain development in a model of mild perinatal asphyxia. Survival rate in the experimental group was 67%. One hour after the insult, intraperitoneally injected Evans blue could be detected in the fetuses’ brains, indicating disruption of the blood-brain barrier. Although brain mass and absolute cell numbers (neurons and non-neurons) were not reduced after perinatal asphyxia immediately and in late brain development, subcellular alterations were detected. Cortical oxygen consumption increased immediately after asphyxia, and remained high up to 7 days, returning to normal levels after 14 days. We observed an increased resistance to mitochondrial membrane permeability transition, and calcium buffering capacity in asphyxiated animals from birth to 14 days after the insult. In contrast to ex vivo data, mitochondrial oxygen consumption in primary cell cultures of neurons and astrocytes was not altered after 1% hypoxia. Taken together, our results demonstrate that although newborns were viable and apparently healthy, brain development is subcellularly altered by perinatal asphyxia. Our findings place the neonate brain mitochondria as a potential target for therapeutic protective interventions.

Image-Based Profiling of Synaptic Connectivity in Primary Neuronal Cell Culture.

Neurological disorders display a broad spectrum of clinical manifestations. Yet, at the cellular level, virtually all these diseases converge into a common phenotype of dysregulated synaptic connectivity. In dementia, synapse dysfunction precedes neurodegeneration and cognitive impairment by several years, making the synapse a crucial entry point for the development of diagnostic and therapeutic strategies. Whereas high-resolution imaging and biochemical fractionations yield detailed insight into the molecular composition of the synapse, standardized assays are required to quickly gauge synaptic connectivity across large populations of cells under a variety of experimental conditions. Such screening capabilities have now become widely accessible with the advent of high-throughput, high-content microscopy. In this review, we discuss how microscopy-based approaches can be used to extract quantitative information about synaptic connectivity in primary neurons with deep coverage. We elaborate on microscopic readouts that may serve as a proxy for morphofunctional connectivity and we critically analyze their merits and limitations. Finally, we allude to the potential of alternative culture paradigms and integrative approaches to enable comprehensive profiling of synaptic connectivity.

Electrical Stimulation, Recording and Impedance-Based Real-Time Position Detection of Cultured Neurons Using Thin-Film-Transistor Array

A novel thin-film-transistor (TFT)-based microsystem array derived from flat panel technology has been developed for functional extracellular neural stimulation and recording. These arrays are microfabricated to meet the needs of optical transparency and extremely high density of electrodes with a large sensor area over 25 mm $\times$ 27 mm. Indium tin oxide has been used for surface electrode fabrication with the pitches of 50 and 100 $\mu \text{m}$ and an optical transparency of ~95%. Each individual electrode is controlled through transistor drain voltage. The array consists of 30000 transistors over 243.75 mm2 chip area. The device has an extra extra high dot per inch (extra extra high density) resolution of 359.2 PPI (pixels per inch) or $7.07\times 10^{-4}$ mm dot pitch. The durability of the stimulated electrodes is extremely high, lasting more than one billion pulses without failure in dry as well as wet environments. Primary neuron cell cultures were established inside the polydimethylsiloxane chamber on the TFT substrate. Extracellular electrical signals were successfully recorded for both the current- and voltage-type stimulations. The binary presence of cells in a given region was achieved by means of impedance measurement. [2017-0266]

Neuroregeneration improved by ketones

Ketone bodies serve as alternative fuel for the brain when glucose availability is limited. The ketone body beta‐hydroxybutyrate (BHB) is synthesized in the liver from acetoacetate, the first ketone produced in the fasting state. Several studies show that being in the state of ketosis can be neuroprotective, but the exact mechanism is not known. It is also not known whether ketones would aid regeneration of a nervous system injury. In this study we investigated on cell cultures whether ketone bodies improve neuroregeneration after injury or trauma.The effect of ketone bodies on cell regeneration was tested in 3 weeks old rat (Sprague Dawley) primary neuronal cell cultures. Scratch assay was performed for 24 hours, while the cells were either exposed to 2mM R, S‐sodium‐3‐hydroxibutyrate (BHB) or not (control). One hundred snapshots were taken with the CYTOSMART system to document the cell migration, changes in cell density and the process of cell regeneration into the damaged area over the 24 hours. Immunofluorescence reaction was performed to label beta III tubulin and synapsin I in the regenerating areas.Cell cultures treated with BHB showed more intense cell migration and regeneration of the damaged area over the 24 hour period. Significantly more DAPI stained cell nuclei were found in the damaged area in cell cultures with 2mM BHB at 20× (p=0.01)and at 10× (p=0.0004) magnification photos as well, and the cell coverage was higher than in control cell cultures. Markers that are associated with flagellar movement, cytoskeletal functions and axonogenesis, synaptogenesis were denser in treated neuronal cultures at the regeneration site as revealed by fluorescence microscopy. These above results show potential applications for ketogenic compounds for treating traumatic brain injury or other injury to the central nervous system.Support or Funding InformationUniversity of South Florida FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

3,3′‐Dichlorobiphenyl (PCB 11) Promotes Dendritic Arborization in Primary Neurons via CREB‐Dependent Mechanisms

PCB 11 is a byproduct of contemporary paint pigment production that has been detected in environmental media around the world, including milk produced for human consumption. We previously reported that PCB 11 enhances dendritic arborization in primary rat neuronal cell cultures. Since increased dendritic arborization, or hyperconnectivity, is a shared characteristic of many neurodevelopmental disorders (NDDs), our findings suggest this chemical poses a risk to the developing brain. The goal of this study was to understand how PCB 11 promotes dendritic growth. We tested canonical mechanisms of PCB developmental neurotoxicity, including signaling mediated by the ryanodine receptor (RyR), aryl hydrocarbon receptor (AhR), thyroid hormone receptor (THR), and cAMP response element binding protein (CREB). [3H]Ryanodine binding assays were used to assess effects of PCB 11 on RyR activity; luciferase‐reporter cell lines, to assess effects on AhR or THR activation. To assess a functional role for these signaling molecules in PCB 11‐mediated dendritic growth, primary neuron‐glia co‐cultures dissociated from neocortices of neonatal Sprague Dawley rats were transfected at 6 days in vitro (DIV) with a MAP2B‐FusRed construct to label the dendritic arbors of individual neurons. At 7 DIV, cultures were pretreated with pharmacological antagonists and then co‐treated for 48 h with PCB 11 or vehicle (0.1% DMSO). Dendritic morphology was quantified in FusRed‐labeled neurons by quantifying the number of dendritic tips and primary dendrites. PCB 11 had no activity at RyR, AhR or THR, and pharmacological antagonism of these receptors had no significant effect on PCB 11‐induced dendritic growth. However, the dendritic promoting effect of PCB 11 was blocked by the selective inhibitor of CREB, 666‐15 (3‐(3‐Aminopropoxy)‐N‐[2‐[[3‐[[(4‐chloro‐2‐hydroxyphenyl)amino]carbonyl]‐2‐naphthalenyl]oxy]ethyl]‐2‐naphthalenecarboxamide), or by co‐transfection with a short hairpin RNA construct specific for CREB. These results indicate that PCB 11 promotes dendritic growth via interaction with CREB or CREB‐dependent mechanism(s), but not directly interfering with known molecular targets of the higher chlorinated PCBs. In light of emerging evidence implicating perturbations of CREB signaling in the etiology of many NDDs, these findings add to the weight of evidence suggesting that PCB 11 poses a threat to the developing human brain.Support or Funding InformationSupported by NIEHS (R01 ES014901 to PJL & INP; T32 ES007059 to SS; P01 ES013661 to HJL) & NICHD (F32 HD088016 to KPK and U54 HD079125).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanisms of neuroprotection against oxidative stress in the anoxia tolerant turtle Trachemys scripta

The detrimental effects of oxidative stress caused by the accumulation of Reactive Oxygen Species (ROS) have been acknowledged as major factors in aging, senescence, and several neurodegenerative conditions including Parkinson's Disease and stroke (ischemia/reperfusion). Mammalian models are extremely susceptible to these stresses and the restoration of oxygen after anoxia causes oxidative damage; however some organisms including the freshwater turtle Trachemys scripta can withstand such conditions without any apparent pathology. T. scripta thus provides us with a model in which we can investigate physiological mechanisms of neuroprotection in anoxia and reoxygenation without the damaging effects that come with oxidative stress.The ability of the turtle to survive these stressors is thought to be related to its high innate levels of antioxidants and its capacity to suppress ROS production. One potential antioxidant mechanism is the Methionine Sulfoxide Reductase System (Msr) that catalyzes the reduction of oxidized Methionine (Met) residues. Methionine residues are easily oxidized amino acids and oxidized Met is associated with decreased protein activity. Msr may also restore function to damaged proteins and it has been shown to scavenge ROS and thus ameliorate cellular damage.The present study investigated the role and regulation of MsrA in turtle primary neuronal cell cultures exposed to 4 hours of anoxia and 4 hours of anoxia followed by 4 hours of reoxygenation. Anoxia and anoxia/reoxygenation increase MsrA mRNA and protein levels, which suggests a protective role under these conditions. MsrA expression was then altered with target specific siRNA; we found that the knock down of MsrA expression resulted in decreased cell viability. As the factors that control MsrA expression in this model are still unknown, we also induced FOXO3a expression by transfecting a FOXO3a plasmid into the cells, as it has been shown to regulate MsrA levels in other animal models. The induction of FOXO3a protected neurons against cell death. Furthermore, treatment of cells with the green tea extract Epigallocatechin gallate (EGCG), which has antioxidant properties, results in increased expression of FOXO3a when the cells are exposed to oxidative stress. Together these results suggest that MsrA may play an important role in protection against anoxia and oxidative stress in the turtle brain, and may prove to be a target to treat diseases of ischemia and oxidative stress.Support or Funding InformationNIH‐NIA Project # 1R15AG033374‐01,FAU FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Perinatal Asphyxia and Brain Development: Mitochondrial Damage Without Anatomical or Cellular Losses

Perinatal asphyxia remains a significant cause of neonatal mortality and is associated with long-term neurodegenerative disorders. In the present study, we evaluated cellular and subcellular damages to brain development in a model of mild perinatal asphyxia. Survival rate in the experimental group was 67%. One hour after the insult, intraperitoneally injected Evans blue could be detected in the fetuses' brains, indicating disruption of the blood-brain barrier. Although brain mass and absolute cell numbers (neurons and non-neurons) were not reduced after perinatal asphyxia immediately and in late brain development, subcellular alterations were detected. Cortical oxygen consumption increased immediately after asphyxia, and remained high up to 7days, returning to normal levels after 14days. We observed an increased resistance to mitochondrial membrane permeability transition, and calcium buffering capacity in asphyxiated animals from birth to 14days after the insult. In contrast to ex vivo data, mitochondrial oxygen consumption in primary cell cultures of neurons and astrocytes was not altered after 1% hypoxia. Taken together, our results demonstrate that although newborns were viable and apparently healthy, brain development is subcellularly altered by perinatal asphyxia. Our findings place the neonate brain mitochondria as a potential target for therapeutic protective interventions.