A primary neural cell culture model to study neuron, astrocyte, and microglia interactions in neuroinflammation.

BackgroundInteractions between neurons, astrocytes, and microglia critically influence neuroinflammatory responses to insult in the central nervous system. In vitro astrocyte and microglia cultures are powerful tools to study specific molecular pathways involved in neuroinflammation; however, in order to better understand the influence of cellular crosstalk on neuroinflammation, new multicellular culture models are required.MethodsPrimary cortical cells taken from neonatal rats were cultured in a serum-free "tri-culture" medium formulated to support neurons, astrocytes, and microglia, or a "co-culture" medium formulated to support only neurons and astrocytes. Caspase 3/7 activity and morphological changes were used to quantify the response of the two culture types to different neuroinflammatory stimuli mimicking sterile bacterial infection (lipopolysaccharide (LPS) exposure), mechanical injury (scratch), and seizure activity (glutamate-induced excitotoxicity). The secreted cytokine profile of control and LPS-exposed co- and tri-cultures were also compared.ResultsThe tri-culture maintained a physiologically relevant representation of neurons, astrocytes, and microglia for 14 days in vitro, while the co-cultures maintained a similar population of neurons and astrocytes, but lacked microglia. The continuous presence of microglia did not negatively impact the overall health of the neurons in the tri-culture, which showed reduced caspase 3/7 activity and similar neurite outgrowth as the co-cultures, along with an increase in the microglia-secreted neurotrophic factor IGF-1 and a significantly reduced concentration of CX3CL1 in the conditioned media. LPS-exposed tri-cultures showed significant astrocyte hypertrophy, increase in caspase 3/7 activity, and the secretion of a number of pro-inflammatory cytokines (e.g., TNF, IL-1α, IL-1β, and IL-6), none of which were observed in LPS-exposed co-cultures. Following mechanical trauma, the tri-culture showed increased caspase 3/7 activity, as compared to the co-culture, along with increased astrocyte migration towards the source of injury. Finally, the microglia in the tri-culture played a significant neuroprotective role during glutamate-induced excitotoxicity, with significantly reduced neuron loss and astrocyte hypertrophy in the tri-culture.ConclusionsThe tri-culture consisting of neurons, astrocytes, and microglia more faithfully mimics in vivo neuroinflammatory responses than standard mono- and co-cultures. This tri-culture can be a useful tool to study neuroinflammation in vitro with improved accuracy in predicting in vivo neuroinflammatory phenomena

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Content protection mechanisms are intended to enforce the usage rights on the content. These usage rights are carried by a license. Sometimes, a license even carries the key that is used to unlock the protected content. Unfortunately, license protection is difficult, yet it is important for digital rights management (DRM). Not many license protection schemes are available, and most if not all are proprietary. In this paper, we present a license protection scheme, which exploits tamper-resistant cryptographic hardware. The confidentiality and integrity of the license or parts thereof can be assured with our protection scheme. In addition, the keys to unlock the protected content are always protected and stored securely as part of the license. We verify secrecy and authentication aspects of one of our protocols. We implement the scheme in a prototype to assess the performance.\ud This project is funded by Telematica Instituut, The Netherlands

Identification and Evolution of Drug Efflux Pump in Clinical Enterobacter aerogenes Strains Isolated in 1995 and 2003

BACKGROUND: The high mortality impact of infectious diseases will increase due to accelerated evolution of antibiotic resistance in important human pathogens. Development of antibiotic resistance is a evolutionary process inducing the erosion of the effectiveness of our arsenal of antibiotics. Resistance is not necessarily limited to a single class of antibacterial agents but may affect many unrelated compounds; this is termed 'multidrug resistance' (MDR). The major mechanism of MDR is the active expulsion of drugs by bacterial pumps; the treatment of gram negative bacterial infections is compromised due to resistance mechanisms including the expression of efflux pumps that actively expel various usual antibiotics (beta-lactams, quinolones, ...). METHODOLOGY/PRINCIPAL FINDINGS: Enterobacter aerogenes has emerged among Enterobacteriaceae associated hospital infections during the last twenty years due to its faculty of adaptation to antibiotic stresses. Clinical isolates of E. aerogenes belonging to two strain collections isolated in 1995 and 2003 respectively, were screened to assess the involvement of efflux pumps in antibiotic resistance. Drug susceptibility assays were performed on all bacterial isolates and an efflux pump inhibitor (PAbetaN) previously characterized allowed to decipher the role of efflux in the resistance. Accumulation of labelled chloramphenicol was monitored in the presence of an energy poison to determine the involvement of active efflux on the antibiotic intracellular concentrations. The presence of the PAbetaN-susceptible efflux system was also identified in resistant E. aerogenes strains. CONCLUSIONS/SIGNIFICANCE: For the first time a noticeable increase in clinical isolates containing an efflux mechanism susceptible to pump inhibitor is report within an 8 year period. After the emergence of extended spectrum beta-lactamases in E. aerogenes and the recent characterisation of porin mutations in clinical isolates, this study describing an increase in inhibitor-susceptible efflux throws light on a new step in the evolution of mechanism in E. aerogenes

Avicin D: A Protein Reactive Plant Isoprenoid Dephosphorylates Stat 3 by Regulating Both Kinase and Phosphatase Activities

Avicins, a class of electrophilic triterpenoids with pro-apoptotic, anti-inflammatory and antioxidant properties, have been shown to induce redox-dependant post-translational modification of cysteine residues to regulate protein function. Based on (a) the cross-talk that occurs between redox and phosphorylation processes, and (b) the role of Stat3 in the process of apoptosis and carcinogenesis, we chose to study the effects of avicins on the processes of phosphorylation/dephosphorylation in Stat3. Avicins dephosphorylate Stat3 in a variety of human tumor cell lines, leading to a decrease in the transcriptional activity of Stat3. The expression of Stat3-regulated proteins such as c-myc, cyclin D1, Bcl2, survivin and VEGF were reduced in response to avicin treatment. Underlying avicin-induced dephosphorylation of Stat3 was dephosphorylation of JAKs, as well as activation of protein phosphatase-1. Downregulation of both Stat3 activity and expression of Stat 3-controlled pro-survival proteins, contributes to the induction of apoptosis in avicin treated tumor cells. Based on the role of Stat3 in inflammation and wounding, and the in vivo inhibition of VEGF by avicins in a mouse skin carcinogenesis model, it is likely that avicin-induced inhibition of Stat3 activity results in the suppression of the pro-inflammatory and pro-oxidant stromal environment of tumors. Activation of PP-1, which also acts as a cellular economizer, combined with the redox regulation by avicins, can aid in redirecting metabolism from growth promoting anabolic to energy sparing pathways

Correction to: A primary neural cell culture model to study neuron, astrocyte, and microglia interactions in neuroinflammation.

Following publication of the original article [1], the authors identified a typo in one value. The 5 μg/mL as the LPS concentration were wrongly given as 5 μM in several instances in the article. This typo does not affect any results or conclusions. It should read as: 5 μg/mL

A Primary Neural Cell Culture Model for Neuroinflammation

AbstractInteractions between neurons, astrocytes and microglia critically influence neuroinflammatory responses to insult in the central nervous system. Studying neuroinflammation in vitro has been difficult because most primary culture models do not include all three critical cell types. We describe an in vitro model of neuroinflammation comprised of neurons, astrocytes and microglia. Primary rat cortical cells were cultured in a serum-free medium used to co-culture neurons and astrocytes that is supplemented with three factors (IL-34, TGF-β and cholesterol) used to support isolated microglia. This “tri-culture” can be maintained for at least 14 days in vitro while retaining a physiologically-relevant representation of all three cell types. Additionally, we demonstrate that the tri-culture system responds to lipopolysaccharide, mechanical trauma and excitotoxicity with both neurotoxic and neuroprotective aspects of the neuroinflammatory response observed in vivo. We expect the tri-culture model will enable mechanistic studies of neuroinflammation in vitro with enhanced physiological relevance

A primary neural cell culture model to study neuron, astrocyte, and microglia interactions in neuroinflammation.

BackgroundInteractions between neurons, astrocytes, and microglia critically influence neuroinflammatory responses to insult in the central nervous system. In vitro astrocyte and microglia cultures are powerful tools to study specific molecular pathways involved in neuroinflammation; however, in order to better understand the influence of cellular crosstalk on neuroinflammation, new multicellular culture models are required.MethodsPrimary cortical cells taken from neonatal rats were cultured in a serum-free "tri-culture" medium formulated to support neurons, astrocytes, and microglia, or a "co-culture" medium formulated to support only neurons and astrocytes. Caspase 3/7 activity and morphological changes were used to quantify the response of the two culture types to different neuroinflammatory stimuli mimicking sterile bacterial infection (lipopolysaccharide (LPS) exposure), mechanical injury (scratch), and seizure activity (glutamate-induced excitotoxicity). The secreted cytokine profile of control and LPS-exposed co- and tri-cultures were also compared.ResultsThe tri-culture maintained a physiologically relevant representation of neurons, astrocytes, and microglia for 14 days in vitro, while the co-cultures maintained a similar population of neurons and astrocytes, but lacked microglia. The continuous presence of microglia did not negatively impact the overall health of the neurons in the tri-culture, which showed reduced caspase 3/7 activity and similar neurite outgrowth as the co-cultures, along with an increase in the microglia-secreted neurotrophic factor IGF-1 and a significantly reduced concentration of CX3CL1 in the conditioned media. LPS-exposed tri-cultures showed significant astrocyte hypertrophy, increase in caspase 3/7 activity, and the secretion of a number of pro-inflammatory cytokines (e.g., TNF, IL-1α, IL-1β, and IL-6), none of which were observed in LPS-exposed co-cultures. Following mechanical trauma, the tri-culture showed increased caspase 3/7 activity, as compared to the co-culture, along with increased astrocyte migration towards the source of injury. Finally, the microglia in the tri-culture played a significant neuroprotective role during glutamate-induced excitotoxicity, with significantly reduced neuron loss and astrocyte hypertrophy in the tri-culture.ConclusionsThe tri-culture consisting of neurons, astrocytes, and microglia more faithfully mimics in vivo neuroinflammatory responses than standard mono- and co-cultures. This tri-culture can be a useful tool to study neuroinflammation in vitro with improved accuracy in predicting in vivo neuroinflammatory phenomena

Influence of microchannel geometry on device performance and electrophysiological recording fidelity during long-term studies of connected neural populations.

Compartmentalized microfluidic neural cell culture platforms, which physically separate axons from the neural soma using a series of microchannels, have been used for studying a wide range of pathological conditions and basic neuroscience questions. While each study has different experimental needs, the fundamental design of these devices has largely remained unchanged and a systematic study to establish long-term neural cultures in this format is lacking. Here, we investigate the influence of microchannel geometry and cell seeding density on device performance particularly in the context of long-term studies of synaptically-connected, yet fluidically-isolated neural populations of neurons and glia. Of the different experimental parameters, the microchannel height was the principal determinant of device performance, where the other parameters offer additional degrees of freedom in customizing such devices for specific applications. We condense the effects of these parameters into design rules and demonstrate their utility in engineering a microfluidic neural culture platform with integrated microelectrode arrays. The engineered device successfully recorded from primary rat cortical cells for 59 days in vitro with more than on order of magnitude enhancement in signal-to-noise ratio in the microchannels

License protection with a tamper-resistant token

Content protection mechanisms are intended to enforce the usage rights on the content. These usage rights are carried by a license. Sometimes, a license even carries the key that is used to unlock the protected content. Unfortunately, license protection is difficult, yet it is important for digital rights management (DRM). Not many license protection schemes are available, and most if not all are proprietary. In this paper, we present a license protection scheme, which exploits tamper-resistant cryptographic hardware. The confidentiality and integrity of the license or parts thereof can be assured with our protection scheme. In addition, the keys to unlock the protected content are always protected and stored securely as part of the license. We verify secrecy and authentication aspects of one of our protocols. We implement the scheme in a prototype to assess the performance