A general method to regenerate arrayed gold microelectrodes for label-free cell assay

We developed a method to regenerate arrayed gold microelectrodes equipped for a commercial label-free cell analyzer. The regeneration process includes efficient treatment of the gold surface with trypsin (0.25%, v/v) digestion, rinsing with ethanol and deionized water and spinning steps. The proposed method ensured complete regeneration and repeated usage of gold microchips up to 4 times for the real-time electric impedance measurement of anti-cancer drug cytotoxicity.     

Neuroprotection of Interleukin-6 Against NMDA-induced Neurotoxicity is Mediated by JAK/STAT3, MAPK/ERK, and PI3K/AKT Signaling Pathways

We have previously shown that interleukin-6 (IL-6) has neuroprotective effect against N-methyl-d-aspartate (NMDA)-induced excitotoxicity. The current study aimed to reveal signal transduction pathways involved in the IL-6 neuroprotection. Cerebellar granule neurons (CGNs) from postnatal 8-day infant rats were exposed to IL-6 (120 ng/ml) for 8 days and stimulated with NMDA (100 μM) for 15 or 30 min. Dynamic intracellular Ca²⁺ fluorescence intensity, cytosolic Ca²⁺-dependent phospholipase A2 (cPLA2) expression, and apoptosis and necrosis in cultured CGNs were measured by laser scanning confocal microscope, real-time PCR and Western blot, and annexin V-FITC/propidium iodide staining, respectively. NMDA stimulation of neurons evoked an intracellular Ca²⁺ overload, an upregulated expression of cPLA2, and an increase in cell death. Chronic IL-6 exposure prevented the NMDA-evoked neuronal Ca²⁺ overload, cPLA2 expression upregulation, and apoptosis and necrosis. Anti-gp130 monoclonal antibody (mAb), a blocker of gp130 that is a 130-kDa signal-transducing β-subunit of IL-6 receptor complex, blocked these effects of IL-6 preventing NMDA neurotoxicity. AG490, PD98059, or LY294002, inhibitors specific for the intracellular signals, JAK, MAPK, and PI3K, respectively, partially blocked these IL-6 neuroprotective effects. Phosphorylation levels of STAT3, ERK1/2, and AKT, the downstream proteins for these enzymes of JAK, MAPK, and PI3K, respectively, were elevated by IL-6 pretreatment. The enhanced activation of STAT3, ERK1/2, and AKT by IL-6 was abolished by AG490, PD98059, and LY294002, respectively. Anti-gp130 mAb attenuated the activation of all the three detected signaling molecules. The present findings suggest that IL-6 neuroprotection is jointly mediated by the cellular signal transduction pathways, gp130-JAK-STAT3, gp130-MAPK-ERK, and gp130-PI3K-AKT.     

Reduced neuroprotective potential of the mesenchymal stromal cell secretome with ex vivo expansion,age and progressive multiple sclerosis

Background

Clinical trials using ex vivo expansion of autologous mesenchymal stromal cells (MSCs) are in progress for several neurological diseases including multiple sclerosis (MS). Given that environment alters MSC function, we examined whether in vitro expansion, increasing donor age and progressive MS affect the neuroprotective properties of the MSC secretome.

Real-Time Monitoring of Photocytotoxicity in Nanoparticles-Based Photodynamic Therapy: A Model-Based Approach

Nanoparticles are widely suggested as targeted drug-delivery systems. In photodynamic therapy (PDT), the use of multifunctional nanoparticles as photoactivatable drug carriers is a promising approach for improving treatment efficiency and selectivity. However, the conventional cytotoxicity assays are not well adapted to characterize nanoparticles cytotoxic effects and to discriminate early and late cell responses. In this work, we evaluated a real-time label-free cell analysis system as a tool to investigate in vitro cyto- and photocyto-toxicity of nanoparticles-based photosensitizers compared with classical metabolic assays. To do so, we introduced a dynamic approach based on real-time cell impedance monitoring and a mathematical model-based analysis to characterize the measured dynamic cell response. Analysis of real-time cell responses requires indeed new modeling approaches able to describe suited use of dynamic models. In a first step, a multivariate analysis of variance associated with a canonical analysis of the obtained normalized cell index (NCI) values allowed us to identify different relevant time periods following nanoparticles exposure. After light irradiation, we evidenced discriminant profiles of cell index (CI) kinetics in a concentration- and light dose-dependent manner. In a second step, we proposed a full factorial design of experiments associated with a mixed effect kinetic model of the CI time responses. The estimated model parameters led to a new characterization of the dynamic cell responses such as the magnitude and the time constant of the transient phase in response to the photo-induced dynamic effects. These parameters allowed us to characterize totally the in vitro photodynamic response according to nanoparticle-grafted photosensitizer concentration and light dose. They also let us estimate the strength of the synergic photodynamic effect. This dynamic approach based on statistical modeling furnishes new insights for in vitro characterization of nanoparticles-mediated effects on cell proliferation with or without light irradiation.     

Insulin-like growth factor-I receptors and estrogen receptors interact in the promotion of neuronal survival and neuroprotection

Several in vitro and in vivo studies have shown that estrogen has neuroprotective properties. The neuroprotective effects of estrogen are probably exerted through several mechanisms. It is established that estrogen can provide neuroprotection by actions that are independent of estrogen receptor activation. In addition, in several experimental models, activation of estrogen receptors appears to be indispensable for neuroprotection. This review focuses on neuroprotection mediated by estrogen receptors. The interaction of estrogen with growth factor receptor signaling to induce neuroprotection is discussed. Evidence is presented that estrogen receptors and insulin-like growth factor-I receptors interact in the promotion of neuronal survival and neuroprotection.     

Phenotypic pattern-based assay for dynamically monitoring host cellular responses to Salmonella infections

The interaction between mammalian host cells and bacteria is a dynamic process, and the underlying pathologic mechanisms are poorly characterized. Limited information describing the host-bacterial interaction is based mainly on studies using label-based endpoint assays that detect changes in cell behavior at a given time point, yielding incomplete information. In this paper, a novel, label-free, real-time cell-detection system based on electronic impedance sensor technology was adapted to dynamically monitor the entire process of intestinal epithelial cells response to Salmonella infection. Changes in cell morphology and attachment were quantitatively and continuously recorded following infection. The resulting impedance-based time-dependent cell response profiles (TCRPs) were compared to standard assays and showed good correlation and sensitivity. Biochemical assays further suggested that TCRPs were correlated with cytoskeleton-associated morphological dynamics, which can be largely attenuated by inhibitions of actin and microtubule polymerization. Collectively, our data indicate that cell-electrode impedance measurements not only provide a novel, real-time, label-free method for investigating bacterial infection but also help advance our understanding of host responses in a more physiological and continuous manner that is beyond the scope of current endpoint assays.     

Postsynaptic scaffold protein Homer 1a protects against traumatic brain injury via regulating group I metabotropic glutamate receptors

Traumatic brain injury (TBI) produces excessive glutamate, leading to excitotoxicity via the activation of glutamate receptors. Postsynaptic density scaffold proteins have crucial roles in mediating signal transduction from glutamate receptors to their downstream mediators. Therefore, studies on the mechanisms underlying regulation of excitotoxicity by scaffold proteins can uncover new treatments for TBI. Here, we demonstrated that the postsynaptic scaffold protein Homer 1a was neuroprotective against TBI in vitro and in vivo, and this neuroprotection was associated with its effects on group I metabotropic glutamate receptors (mGluRs). Upon further study, we found that Homer 1a mainly affected neuronal injury induced by mGluR1 activation after TBI and also influenced mGluR5 function when its activity was restored. The ability of Homer 1a to disrupt mGluR-ERK signaling contributed to its ability to regulate the functions of mGluR1 and mGluR5 after traumatic injury. Intracellular Ca²⁺ and PKC were two important factors involved in the mediation of mGluR-ERK signaling by Homer 1a. These results define Homer 1a as a novel endogenous neuroprotective agent against TBI.     

Dynamic assessment of cell viability,proliferation and migration using real time cell analyzer system (RTCA)

Cell viability and cell migration capacities are critical parameters for cell culture-related studies. It is essential to monitor the dynamic changes of cell properties under various co-culture conditions to our better understanding of their behaviours and characteristics. The real time cell analyzer (RTCA, xCELLigence, Roche) is an impedance-based technology that can be used for label-free and real-time monitoring of cell properties, such as cell adherence, proliferation, migration and cytotoxicity. The practicality of this system has been proven in our recent cancer studies. In the present method, we intend to use co-cultures of pancreatic cancer cells (HP62) and mesenchymal stem cells to describe in detail, the procedures and benefits of RTCA.     

Carvedilol,a third-generation β-blocker prevents oxidative stress-induced neuronal death and activates Nrf2/ARE pathway in HT22 cells

Carvedilol, a nonselective β-adrenoreceptor blocker with pleiotropic activities has been shown to exert neuroprotective effect due to its antioxidant property. However, the neuroprotective mechanism of carvedilol is still not fully uncovered. Nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is an important cellular stress response pathway involved in neuroprotection. Here we investigated the effect of carvedilol on oxidative stress-induced cell death (glutamate 2 mM and H₂O₂ 600 μM) and the activity of Nrf2/ARE pathway in HT22 hippocampal cells. Carvedilol significantly increased cell viability and decreased ROS in HT22 cells exposed to glutamate or H₂O₂. Furthermore, carvedilol activated the Nrf2/ARE pathway in a concentration-dependent manner, and increased the protein levels of heme oxygenase-1(HO-1) and NAD(P)H quinone oxidoreductase-1(NQO-1), two downstream factors of the Nrf2/ARE pathway. Collectively, our results indicate that carvedilol protects neuronal cell against glutamate- and H₂O₂-induced neurotoxicity possibly through activating the Nrf2/ARE signaling pathway.     

Dynamic Monitoring of Cellular Remodeling Induced by the Transforming Growth Factor-β1

The plasticity of differentiated adult cells could have a great therapeutic potential, but at the same time, it is characteristic of progression of serious pathological states such as cancer and fibrosis. In this study, we report on the application of a real-time noninvasive system for dynamic monitoring of cellular plasticity. Analysis of the cell impedance profile recorded as cell index using a real-time cell analyzer revealed its significant increase after the treatment of prostate epithelial cells with the transforming growth factor-β1. Changes in the cell index profile were paralleled with cytoskeleton rebuilding and induction of epithelial–mesenchymal transition and negatively correlated with cell proliferation. This novel application of such approach demonstrated a great potential of the impedance-based system for noninvasive and real-time monitoring of cellular fate.     

Paeoniflorin, a Natural Neuroprotective Agent, Modulates Multiple Anti-Apoptotic and Pro-apoptotic Pathways in Differentiated PC12 Cells

Numerous studies have shown robust neuroprotective effects of paeoniflorin (PF), a natural compound derived from the herbal medicine Paeony radix. In the present study, we determined associations of PF neuroprotection with its modulation of various apoptotic and anti-apoptotic pathways. PF (50–400 μM) pretreatment significantly improved viability of differentiated PC12 cells exposed to methyl-4-phenylpyridine ion (MPP⁺), a neurotoxin, and inhibited over-release of lactate dehydrogenase, a biomarker of neuronal cell death. PF also ameliorated MPP⁺-induced nuclear and mitochondrial apoptotic alteration and intracellular calcium overload. PF treatment reversed MPP⁺ suppression of activity of B cell lymphoma-extra large, which is a mitochondrial membrane molecule that protects cells from DNA damage-induced apoptosis, and strikingly inhibited the enhanced level of cleaved poly(ADP-ribose)polymerase, which is involved in the process of apoptosis. PF alone and coadministration with MPP⁺ enhanced phospho activation of extracellular signal-regulated kinases, Akt, and its downstream element glycogen synthase kinase-3, but the effects were completely abolished in the presence of their blockers PD98059 and LY294002. The presence of the blockers also diminished the potency of PF in improving viability of MPP⁺-exposed cells. These results indicate that neuroprotective effects of PF are related to its modulation of multiple anti-apoptotic and pro-apoptotic pathways, including blockade of intracellular calcium overload, prevention of mitochondrial membrane integrity, inhibition of pro-apoptotic molecules, and up-regulation of anti-apoptotic proteins associated with cell survival and proliferation. The study provides evidence supporting PF as a potential therapeutic agent used for the treatment of neurodegenerative diseases and neural injury.     

Luteolin provides neuroprotection in models of traumatic brain injury via the Nrf2–ARE pathway

Luteolin has recently been proven to exert neuroprotection in a variety of neurological diseases; however, its roles and the underlying mechanisms in traumatic brain injury are not fully understood. The present study was aimed to investigate the neuroprotective effects of luteolin in models of traumatic brain injury (TBI) and the possible role of the Nrf2–ARE pathway in the putative neuroprotection. A modified Marmarou׳s weight-drop model in mice and the scratch model in mice primary cultured neurons were used to induce TBI. We determined that luteolin significantly ameliorated secondary brain injury induced by TBI, including neurological deficits, brain water content, and neuronal apoptosis. Furthermore, the level of malondialdehyde (MDA) and the activity of glutathione peroxidase (GPx) were restored in the group with luteolin treatment. in vitro studies showed that luteolin administration lowered the intracellular reactive oxygen species (ROS) level and increased the neuron survival. Moreover, luteolin enhanced the translocation of Nrf2 to the nucleus both in vivo and in vitro, which was proved by the results of Western blot, immunohistochemistry, and electrophoretic mobility shift assay (EMSA). Subsequently upregulation of the expression of the downstream factors such as heme oxygenase 1 (HO1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) was also examined. However, luteolin treatment failed to provide neuroprotection after TBI in Nrf2^-/- mice. Taken together, these in vivo and in vitro data demonstrated that luteolin provided neuroprotective effects in the models of TBI, possibly through the activation of the Nrf2–ARE pathway.     

Protective Effects of Paeoniflorin Against Glutamate-Induced Neurotoxicity in PC12 Cells via Antioxidant Mechanisms and Ca<Superscript>2+</Superscript> Antagonism

Preclinical and clinical investigations have shown hippocampal neuronal atrophy and destruction were observed in patients with depression, which could be ameliorated by the treatment with antidepressants. Therefore, neuroprotection has been proposed to be one of the acting mechanisms of antidepressant. Paeoniflorin, a monoterpene glycoside, has been reported to display antidepressant-like effects in animal models of behavioral despair. The present study aimed to examine the protective effect of paeoniflorin on glutamate-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. The results showed that pretreatment with paeoniflorin elevated cell viability, inhibited apoptosis, decreased levels of intracellular reactive oxygen species and malondialdehyde, and enhanced activity of superoxide dismutase in glutamate-treated PC12 cells. Pretreatment with paeoniflorin also reversed the increased intracellular Ca²⁺ concentration and the reduced Calbindin-D28K mRNA level caused by glutamate in PC12 cells. The results suggest that paeoniflorin exerts a neuroprotective effect on glutamate-induced neurotoxicity in PC12 cells, at least in part, via inhibiting oxidative stress and Ca²⁺ overload. This neuroprotective effect may be one of the action pathways accounting for the in vivo antidepressant activity of paeoniflorin.     

Analysis of inter-/intra-E-plate repeatability in the real-time cell analyzer

Over the past decade, the real-time cell analyzer (RTCA) has provided a good tool to the cell-based in vitro assay. Unlike the traditional systems that label the target cells with luminescence, fluorescence, or light absorption, RTCA monitors cell properties using noninvasive and label-free impedance measuring. However, realization of the maximum value of RTCA for applications will require assurance of within-experiment repeatability, day-to-day repeatability, and robustness to variations in conditions that might occur from different experiments. In this article, the performance and variability of RTCA is evaluated and a novel repeatability index (RI) is proposed to analyze the intra-/inter-E-plate repeatability of RTCA. The repeatability assay involves six cell lines and two media (water [H₂O] and dimethyl sulfoxide [DMSO]). First, six cell lines are exposed to the media individually, and time-dependent cellular response curves characterized as a cell index (CI) are recorded by RTCA. Then, the variations along sampling time and among repeated tests are calculated and RI values are obtained. Finally, a discriminating standard is set up to evaluate the degree of repeatability. As opposed to the standardized methodologies, it is shown that the presented index can give the quantitative evaluation for repeatability of RTCA within E-plate and variation on different days.     

Involvement of α7 nAChR Signaling Cascade in Epigallocatechin Gallate Suppression of β-Amyloid-Induced Apoptotic Cortical Neuronal Insults

Excessive generation and accumulation of the β-amyloid (Aβ) peptide in selectively vulnerable brain regions is a key pathogenic event in the Alzheimer's disease (AD), while epigallocatechin gallate (EGCG) is a very promising chemical to suppress a variety of Aβ-induced neurodegenerative disorders. However, the precise molecular mechanism of EGCG responsible for protection against neurotoxicity still remains elusive. To validate and further investigate the possible mechanism involved, we explored whether EGCG neuroprotection against neurotoxicity of Aβ is mediated through the α7 nicotinic acetylcholine receptor (α7 nAChR) signaling cascade. It was shown in rat primary cortical neurons that short-term treatment with EGCG significantly attenuated the neurotoxicity of Aβ_1–42, as demonstrated by increased cell viability, reduced number of apoptotic cells, decreased reactive oxygen species (ROS) generation, and downregulated caspase-3 levels after treatment with 25-μM Aβ_1–42. In addition, EGCG markedly strengthened activation of α7nAChR as well as its downstream pathway signaling molecules phosphatidylinositol 3-kinase (PI3K) and Akt, subsequently leading to suppression of Bcl-2 downregulation in Aβ-treated neurons. Conversely, administration of α7nAChR antagonist methyllycaconitine (MLA; 20 μM) to neuronal cultures significantly attenuated the neuroprotection of EGCG against Aβ-induced neurototoxicity, thus presenting new evidence that the α7nAChR activity together with PI3K/Akt transduction signaling may contribute to the molecular mechanism underlying the neuroprotective effects of EGCG against Aβ-induced cell death.     

Protection from MPTP-induced neurotoxicity in differentiating mouse N2a neuroblastoma cells

We have shown previously that subcytotoxic concentrations of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) inhibit axon outgrowth and are associated with increased neurofilament heavy chain (NF-H) phosphorylation in differentiating mouse N2a neuroblastoma cells while higher doses (> 100 microM) cause cell death. In this work we assessed the ability of potential neuroprotective agents to alleviate both MPTP-induced cell death (cytotoxicity) and MPTP-induced NF-H phosphorylation/reduction in axon outgrowth (neurotoxicity) in N2a cells induced to differentiate by dbcAMP. The neurotoxic effects of MPTP occurred in the absence of significant alterations in energy status or mitochondrial membrane potential. The hormone oestradiol (100 microM) reduced the cytotoxic effect of MPTP, but blocked di-butyryl cyclic AMP (dbcAMP)-induced differentiation, i.e. axon outgrowth. Both the cytotoxic and neurotoxic effects of MPTP were reduced by the monoamine oxidase (MAO) inhibitors deprenyl and, to a lesser extent, clorgyline. Alleviation of both neurotoxicity and cytotoxicity was also achieved by conditioned medium derived from rat C6 glioma cells. In contrast, whilst the p38 MAP kinase inhibitor, SB202190, protected cells against MPTP-induced neurotoxicity, it could not maintain cell viability at high MPTP exposures. In each case neuroprotection involved maintenance of the differentiating phenotype linked with attenuation of NF-H hyper-phosphorylation; the latter may represent a mechanism by which neuronal cells can moderate MPTP-induced neurotoxicity. The use of a simplified neuronal cell model, which expresses subtle biochemical changes following neurotoxic insult, could therefore provide a valuable tool for the identification of potential neuroprotective agents.     

The use of real-time cell analyzer technology in drug discovery: defining optimal cell culture conditions and assay reproducibility with different adherent cellular models

The use of impedance-based label-free technology applied to drug discovery is nowadays receiving more and more attention. Indeed, such a simple and noninvasive assay that interferes minimally with cell morphology and function allows one to perform kinetic measurements and to obtain information on proliferation, migration, cytotoxicity, and receptor-mediated signaling. The objective of the study was to further assess the usefulness of a real-time cell analyzer (RTCA) platform based on impedance in the context of quality control and data reproducibility. The data indicate that this technology is useful to determine the best coating and cellular density conditions for different adherent cellular models including hepatocytes, cardiomyocytes, fibroblasts, and hybrid neuroblastoma/neuronal cells. Based on 31 independent experiments, the reproducibility of cell index data generated from HepG2 cells exposed to DMSO and to Triton X-100 was satisfactory, with a coefficient of variation close to 10%. Cell index data were also well reproduced when cardiomyocytes and fibroblasts were exposed to 21 compounds three times (correlation >0.91, p < 0.0001). The data also show that a cell index decrease is not always associated with cytotoxicity effects and that there are some confounding factors that can affect the analysis. Finally, another drawback is that the correlation analysis between cellular impedance measurements and classical toxicity endpoints has been performed on a limited number of compounds. Overall, despite some limitations, the RTCA technology appears to be a powerful and reliable tool in drug discovery because of the reasonable throughput, rapid and efficient performance, technical optimization, and cell quality control.     

Leukotriene receptor antagonists,LY293111 and ONO-1078, protect neurons from the sPLA2-IB-induced neuronal cell death independently of blocking their receptors

In the ischemic brain, leukotrienes (LTs) are increased and their receptor antagonists protect neurons. However, it has not yet been sufficiently clarified how antagonists for LT receptors exhibit neuroprotective effects. In the present study, we evaluated protective effects of receptor antagonists for LTB₄ (LY293111) and cysteinyl LTs (ONO-1078) in the primary culture of rat cortical neurons. The group IB secretory phospholipase A₂ (sPLA₂-IB)-induced neuronal cell death had been established as the in vitro model for cerebral ischemia. sPLA₂-IB triggered the influx of Ca²⁺ into neurons via L-type voltage-dependent calcium channel (L-VDCC). Subsequently, the enzyme produced eicosanoids including LTB₄ before neuronal cell death. Neither administration of LTB₄ nor cysteinyl LTs such as LTC₄, LTD₄ and LTE₄ killed neurons. However, both LY293111 and ONO-1078 significantly prevented neurons from the neurotoxicity of sPLA₂-IB, suggesting that the two LT receptor blockers protected neurons through alternative pathways beside LT receptors. An L-VDCC blocker does not only inhibit the influx of Ca²⁺ into neurons but also rescues neurons from the sPLA₂-IB-induced neuronal cell death. The two LT receptor antagonists also blocked the sPLA₂-IB-induced Ca²⁺ influx significantly. Thus, LTs exhibited no neurotoxicity, but their receptor antagonists protected neurons directly in the in vitro ischemic model. Furthermore, the suppression of L-VDCC appeared to be involved in the neuroprotective effects of LY293111 and ONO-1078 independent of blocking their receptors.     

A Rac1/phosphatidylinositol 3-kinase/Akt3 anti-apoptotic pathway, triggered by AlsinLF, the product of the ALS2 gene, antagonizes Cu/Zn-superoxide dismutase (SOD1) mutant-induced motoneuronal cell death

AlsinLF, the product of the ALS2 gene, inhibits Cu/Zn-superoxide dismutase (SOD1) mutant-induced neurotoxicity via its Rho guanine nucleotide-exchanging factor domain. We here identified Rac1, a Rho family small GTPase, as a target for the Rho guanine nucleotide-exchanging factor activity of alsinLF. Rac1 associates with alsinLF. The amount of the GTP form of Rac1 is up-regulated by enforced overexpression of alsinLF. We further found not only that constitutively active Rac1 suppresses motoneuronal cell death induced by SOD1 mutants but also that the neuroprotective activity of alsinLF was completely inhibited by knocking down the endogenous Rac1 expression with small interfering RNA for Rac1, indicating that Rac1 is the major effector for alsinLF-mediated neuroprotection. Such alsinLF/Rac1-mediated neuroprotection occurs specifically against the SOD1 mutant-induced cell death but not against the cell death induced by any other neurotoxic insults in motoneuronal NSC34 cells. We further demonstrated that the alsinLF/Rac1-mediated neuroprotective signal is transmitted to the phosphatidylinositol 3-kinase/Akt anti-apoptotic axis. Among three Akt family proteins, Akt3 is the major downstream mediator for alsinLF/Rac1-mediated neuroprotection, which is specifically effective against SOD1 mutant-induced neurotoxicity.