Substrate Topography Determines Neuronal Polarization and Growth In Vitro

The establishment of neuronal connectivity depends on the correct initial polarization of the young neurons. In vivo, developing neurons sense a multitude of inputs and a great number of molecules are described that affect their outgrowth. In vitro, many studies have shown the possibility to influence neuronal morphology and growth by biophysical, i.e. topographic, signaling. In this work we have taken this approach one step further and investigated the impact of substrate topography in the very early differentiation stages of developing neurons, i.e. when the cell is still at the round stage and when the first neurite is forming. For this purpose we fabricated micron sized pillar structures with highly reproducible feature sizes, and analyzed neurons on the interface of flat and topographic surfaces. We found that topographic signaling was able to attract the polarization markers of mouse embryonic neurons -N-cadherin, Golgi-centrosome complex and the first bud were oriented towards topographic stimuli. Consecutively, the axon was also preferentially extending along the pillars. These events seemed to occur regardless of pillar dimensions in the range we examined. However, we found differences in neurite length that depended on pillar dimensions. This study is one of the first to describe in detail the very early response of hippocampal neurons to topographic stimuli.     

Combining Voltage and Calcium Imaging from Neuronal Dendrites

The ability to monitor membrane potential (V _m) and calcium (Ca²⁺) transients at multiple locations on the same neuron can facilitate further progress in our understanding of neuronal function. Here we describe a method to combine V _m and Ca²⁺ imaging using styryl voltage sensitive dyes and Fura type UV-excitable Ca²⁺ indicators. In all cases V _m optical signals are linear with membrane potential changes, but the calibration of optical signals on an absolute scale is presently possible only in some neurons. The interpretation of Ca²⁺ optical signals depends on the indicator Ca²⁺ buffering capacity relative to the cell endogenous buffering capacity. In hippocampal CA1 pyramidal neurons, loaded with JPW-3028 and 300 μM Bis-Fura-2, V _m optical signals cannot be calibrated and the physiological Ca²⁺ dynamics are compromised by the presence of the indicator. Nevertheless, at each individual site, relative changes in V _m and Ca²⁺ fluorescence signals under different conditions can provide meaningful new information on local dendritic integration. In cerebellar Purkinje neurons, loaded with JPW-1114 and 1 mM Fura-FF, V _m optical signals can be calibrated in terms of mV and Ca²⁺ optical signals quantitatively reveal the physiological changes in free Ca²⁺. Using these two examples, the method is explained in detail.     

Dopamine Receptor Activation Reorganizes Neuronal Ensembles during Hippocampal Sharp Waves In Vitro

Hippocampal sharp wave (SW)/ripple complexes are thought to contribute to memory consolidation. Previous studies suggest that behavioral rewards facilitate SW occurrence in vivo. However, little is known about the precise mechanism underlying this enhancement. Here, we examined the effect of dopaminergic neuromodulation on spontaneously occurring SWs in acute hippocampal slices. Local field potentials were recorded from the CA1 region. A brief (1 min) treatment with dopamine led to a persistent increase in the event frequency and the magnitude of SWs. This effect lasted at least for our recording period of 45 min and did not occur in the presence of a dopamine D₁/D₅ receptor antagonist. Functional multineuron calcium imaging revealed that dopamine-induced SW augmentation was associated with an enriched repertoire of the firing patterns in SW events, whereas the overall tendency of individual neurons to participate in SWs and the mean number of cells participating in a single SW were maintained. Therefore, dopaminergic activation is likely to reorganize cell assemblies during SWs.     

Thy1-GCaMP6 Transgenic Mice for Neuronal Population Imaging In Vivo

Genetically-encoded calcium indicators (GECIs) facilitate imaging activity of genetically defined neuronal populations in vivo. The high intracellular GECI concentrations required for in vivo imaging are usually achieved by viral gene transfer using adeno-associated viruses. Transgenic expression of GECIs promises important advantages, including homogeneous, repeatable, and stable expression without the need for invasive virus injections. Here we present the generation and characterization of transgenic mice expressing the GECIs GCaMP6s or GCaMP6f under the Thy1 promoter. We quantified GCaMP6 expression across brain regions and neurons and compared to other transgenic mice and AAV-mediated expression. We tested three mouse lines for imaging in the visual cortex in vivo and compared their performance to mice injected with AAV expressing GCaMP6. Furthermore, we show that GCaMP6 Thy1 transgenic mice are useful for long-term, high-sensitivity imaging in behaving mice.     

Oral Community Interactions of Filifactor alocis In Vitro

Filifactor alocis is a gram positive anaerobe that is emerging as an important periodontal pathogen. In the oral cavity F. alocis colonizes polymicrobial biofilm communities; however, little is known regarding the nature of the interactions between F. alocis and other oral biofilm bacteria. Here we investigate the community interactions of two strains of F. alocis with Streptococcus gordonii, Fusobacterium nucleatum, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, organisms with differing pathogenic potential in the oral cavity. In an in vitro community development model, S. gordonii was antagonistic to the accumulation of F. alocis into a dual species community. In contrast, F. nucleatum and the type strain of F. alocis formed a synergistic partnership. Accumulation of a low passage isolate of F. alocis was also enhanced by F. nucleatum. In three species communities of S. gordonii, F. nucleatum and F. alocis, the antagonistic effects of S. gordonii superseded the synergistic effects of F. nucleatum toward F. alocis. The interaction between A. actinomycetemcomitans and F. alocis was strain specific and A. actinomycetemcomitans could either stimulate F. alocis accumulation or have no effect depending on the strain. P. gingivalis and F. alocis formed heterotypic communities with the amount of P. gingivalis greater than in the absence of F. alocis. However, while P. gingivalis benefited from the relationship, levels of F. alocis in the dual species community were lower compared to F. alocis alone. The inhibitory effect of P. gingivalis toward F. alocis was dependent, at least partially, on the presence of the Mfa1 fimbrial subunit. In addition, AI-2 production by P. gingivalis helped maintain levels of F. alocis. Collectively, these results show that the pattern of F. alocis colonization will be dictated by the spatial composition of microbial microenvironments, and that the organism may preferentially accumulate at sites rich in F. nucleatum.     

Stimulus-Specific Adaptation at the Synapse Level In Vitro

Stimulus-specific adaptation (SSA) is observed in many brain regions in humans and animals. SSA of cortical neurons has been proposed to accumulate through relays in ascending pathways. Here, we examined SSA at the synapse level using whole-cell patch-clamp recordings of primary cultured cortical neurons of the rat. First, we found that cultured neurons had high firing capability with 100-Hz current injection. However, neuron firing started to adapt to repeated electrically activated synaptic inputs at 10 Hz. Next, to activate different dendritic inputs, electrical stimulations were spatially separated. Cultured neurons showed similar SSA properties in the oddball stimulation paradigm compared to those reported in vivo. Single neurons responded preferentially to a deviant stimulus over repeated, standard stimuli considering both synapse-driven spikes and excitatory postsynaptic currents (EPSCs). Compared with two closely placed stimulating electrodes that activated highly overlapping dendritic fields, two separately placed electrodes that activated less overlapping dendritic fields elicited greater SSA. Finally, we used glutamate puffing to directly activate postsynaptic glutamate receptors. Neurons showed SSA to two separately placed puffs repeated at 10 Hz. Compared with EPSCs, GABAa receptor-mediated inhibitory postsynaptic currents showed weaker SSA. Heterogeneity of the synaptic inputs was critical for producing SSA, with glutamate receptor desensitization participating in the process. Our findings suggest that postsynaptic fatigue contributes largely to SSA at low frequencies.     

A Novel and Efficient Gene Transfer Strategy Reduces Glial Reactivity and Improves Neuronal Survival and Axonal Growth In Vitro

Background

The lack of axonal regeneration in the central nervous system is attributed among other factors to the formation of a glial scar. This cellular structure is mainly composed of reactive astrocytes that overexpress two intermediate filament proteins, the glial fibrillary acidic protein (GFAP) and vimentin. Indeed, in vitro, astrocytes lacking GFAP or both GFAP and vimentin were shown to be the substrate for increased neuronal plasticity. Moreover, double knockout mice lacking both GFAP and vimentin presented lower levels of glial reactivity in vivo, significant axonal regrowth and improved functional recovery in comparison with wild-type mice after spinal cord hemisection. From these results, our objective was to develop a novel therapeutic strategy for axonal regeneration, based on the targeted suppression of astroglial reactivity and scarring by lentiviral-mediated RNA-interference (RNAi).

Methods and Findings

In this study, we constructed two lentiviral vectors, Lv-shGFAP and Lv-shVIM, which allow efficient and stable RNAi-mediated silencing of endogenous GFAP or vimentin in vitro. In cultured cortical and spinal reactive astrocytes, the use of these vectors resulted in a specific, stable and highly significant decrease in the corresponding protein levels. In a second model — scratched primary cultured astrocytes — Lv-shGFAP, alone or associated with Lv-shVIM, decreased astrocytic reactivity and glial scarring. Finally, in a heterotopic coculture model, cortical neurons displayed higher survival rates and increased neurite growth when cultured with astrocytes in which GFAP and vimentin had been invalidated by lentiviral-mediated RNAi.

Conclusions

Lentiviral-mediated knockdown of GFAP and vimentin in astrocytes show that GFAP is a key target for modulating reactive gliosis and monitoring neuron/glia interactions. Thus, manipulation of reactive astrocytes with the Lv-shGFAP vector constitutes a promising therapeutic strategy for increasing glial permissiveness and permitting axonal regeneration after central nervous system lesions.     

Perlecan Maintains Microvessel Integrity In Vivo and Modulates Their Formation In Vitro

Perlecan is a heparan sulfate proteoglycan assembled into the vascular basement membranes (BMs) during vasculogenesis. In the present study we have investigated vessel formation in mice, teratomas and embryoid bodies (EBs) in the absence of perlecan. We found that perlecan was dispensable for blood vessel formation and maturation until embryonic day (E) 12.5. At later stages of development 40% of mutant embryos showed dilated microvessels in brain and skin, which ruptured and led to severe bleedings. Surprisingly, teratomas derived from perlecan-null ES cells showed efficient contribution of perlecan-deficient endothelial cells to an apparently normal tumor vasculature. However, in perlecan-deficient EBs the area occupied by an endothelial network and the number of vessel branches were significantly diminished. Addition of FGF-2 but not VEGF₁₆₅ rescued the in vitro deficiency of the mutant ES cells. Furthermore, in the absence of perlecan in the EB matrix lower levels of FGFs are bound, stored and available for cell surface presentation. Altogether these findings suggest that perlecan supports the maintenance of brain and skin subendothelial BMs and promotes vasculo- and angiogenesis by modulating FGF-2 function.     

Characterization of Biofilm Formation by Borrelia burgdorferi In Vitro

Borrelia burgdorferi, the causative agent of Lyme disease, has long been known to be capable of forming aggregates and colonies. It was recently demonstrated that Borrelia burgdorferi aggregate formation dramatically changes the in vitro response to hostile environments by this pathogen. In this study, we investigated the hypothesis that these aggregates are indeed biofilms, structures whose resistance to unfavorable conditions are well documented. We studied Borrelia burgdorferi for several known hallmark features of biofilm, including structural rearrangements in the aggregates, variations in development on various substrate matrices and secretion of a protective extracellular polymeric substance (EPS) matrix using several modes of microscopic, cell and molecular biology techniques. The atomic force microscopic results provided evidence that multilevel rearrangements take place at different stages of aggregate development, producing a complex, continuously rearranging structure. Our results also demonstrated that Borrelia burgdorferi is capable of developing aggregates on different abiotic and biotic substrates, and is also capable of forming floating aggregates. Analyzing the extracellular substance of the aggregates for potential exopolysaccharides revealed the existence of both sulfated and non-sulfated/carboxylated substrates, predominately composed of an alginate with calcium and extracellular DNA present. In summary, we have found substantial evidence that Borrelia burgdorferi is capable of forming biofilm in vitro. Biofilm formation by Borrelia species might play an important role in their survival in diverse environmental conditions by providing refuge to individual cells.     

Amygdalin Influences Bladder Cancer Cell Adhesion and Invasion In Vitro

The cyanogenic diglucoside amygdalin, derived from Rosaceae kernels, is employed by many patients as an alternative anti-cancer treatment. However, whether amygdalin indeed acts as an anti-tumor agent is not clear. Metastasis blocking properties of amygdalin on bladder cancer cell lines was, therefore, investigated. Amygdalin (10 mg/ml) was applied to UMUC-3, TCCSUP or RT112 bladder cancer cells for 24 h or for 2 weeks. Tumor cell adhesion to vascular endothelium or to immobilized collagen as well as tumor cell migration was examined. Effects of drug treatment on integrin α and β subtypes, on integrin-linked kinase (ILK) and total and activated focal adhesion kinase (FAK) were also determined. Integrin knock-down was carried out to evaluate integrin influence on migration and adhesion. A 24 h or 2 week amygdalin application distinctly reduced tumor cell adhesion and migration of UMUC-3 and RT112 cells. TCCSUP adhesion was also reduced, but migration was elevated under amygdalin. Integrin subtype expression was significantly and specifically altered by amygdalin depending on the cell line. ILK was moderately, and activated FAK strongly, lost in all tumor cell lines in the presence of amygdalin. Knock down of β1 integrin caused a significant decrease in both adhesion and migration of UMUC-3 cells, but a significant increase in TCCSUP adhesion. Knock down of β4 integrin caused a significant decrease in migration of RT112 cells. Since the different actions of amygdalin on the different cell lines was mirrored by β1 or β4 knock down, it is postulated that amygdalin influences adhesion and migratory properties of bladder cancer cells by modulating β1 or β4 integrin expression. The amygdalin induced increase in TCCSUP migratory behavior indicates that any anti-tumor benefits from amygdalin (seen with the other two cell lines) may depend upon the cancer cell type.     

Epithelial Transport of Immunogenic and Toxic Gliadin Peptides In Vitro

Scope

Celiac disease is an autoimmune disorder caused by failure of oral tolerance against gluten in genetically predisposed individuals. The epithelial translocation of gluten-derived gliadin peptides is an important pathogenetic step; the underlying mechanisms, however, are poorly understood. Thus, we investigated the degradation and epithelial translocation of two different gliadin peptides, the toxic P31–43 and the immunogenic P56–68. As the size, and hence, the molecular weight of peptides might have an effect on the transport efficiency we chose two peptides of the same, rather short chain length.

Methods and Results

Fluorescence labeled P31–43 and P56–68 were synthesized and studied in a transwell system with human enterocytes. Fluorometric measurements were done to reveal antigen translocation and flow cytometry as well as confocal microscopy were used to investigate cellular uptake of peptides. Structural changes of these peptides were analysed by MALDI-TOF-MS. According to fluorescence intensities, significantly more P31–43 compared to P56–68 was transported through the enterocyte layer after 24 h incubation. In contrast to previous reports, however, mass spectrometric data do not only show a time-dependent cleavage of the immunogenic P56–68, but we observed for the first time the degradation of the toxic peptide P31–43 at the apical side of epithelial cells.

Conclusion

Considering the degradation of gliadin peptides by enterocytes, measurement of fluorescence signals do not completely represent translocated intact gliadin peptides. From our experiments it is obvious that even short peptides can be digested prior to the translocation across the epithelial barrier. Thus, the chain length and the sensibility to degradations of gliadin peptides as well as the integrity of the epithelial barrier seem to be critical for the uptake of gliadin peptides and the subsequent inflammatory immune response.     

Loss of Gadkin Affects Dendritic Cell Migration In Vitro

Migration is crucial for the function of dendritic cells (DCs), which act as outposts of the immune system. Upon detection of pathogens, skin- and mucosa-resident DCs migrate to secondary lymphoid organs where they activate T cells. DC motility relies critically on the actin cytoskeleton, which is regulated by the actin-related protein 2/3 (ARP2/3) complex, a nucleator of branched actin networks. Consequently, loss of ARP2/3 stimulators and upstream Rho family GTPases dramatically impairs DC migration. However, nothing is known yet about the relevance of ARP2/3 inhibitors for DC migration. We previously demonstrated that the AP-1-associated adaptor protein Gadkin inhibits ARP2/3 by sequestering it on intracellular vesicles. Consistent with a role of Gadkin in DC physiology, we here report Gadkin expression in bone marrow-derived DCs and show that its protein level and posttranslational modification are regulated upon LPS-induced DC maturation. DCs derived from Gadkin-deficient mice were normal with regards to differentiation and maturation, but displayed increased actin polymerization. While the actin-dependent processes of macropinocytosis and cell spreading were not affected, loss of Gadkin significantly impaired DC migration in vitro, however, in vivo DC migration was unperturbed suggesting the presence of compensatory mechanisms.     

High Sensitivity of Giardia duodenalis to Tetrahydrolipstatin (Orlistat) In Vitro

Giardiasis, a gastrointestinal disease caused by Giardia duodenalis, is currently treated mainly with nitroimidazoles, primarily metronidazole (MTZ). Treatment failure rates of up to 20 percent reflect the compelling need for alternative treatment options. Here, we investigated whether orlistat, a drug approved to treat obesity, represents a potential therapeutic agent against giardiasis. We compared the growth inhibitory effects of orlistat and MTZ on a long-term in vitro culture adapted G. duodenalis strain, WB-C6, and on a new isolate, 14-03/F7, from a patient refractory to MTZ treatment using a resazurin assay. The giardiacidal concentration of the drugs and their combined in vitro efficacy was determined by median-effect analysis. Morphological changes after treatment were analysed by light and electron microscopy. Orlistat inhibited the in vitro growth of G. duodenalis at low micromolar concentrations, with isolate 14-03/F7 (IC50_24h = 2.8 µM) being more sensitive than WB-C6 (IC50_24h = 6.2 µM). The effect was significantly more potent compared to MTZ (IC50_24h = 4.3 µM and 11.0 µM, respectively) and led to specific undulated morphological alterations on the parasite surface. The giardiacidal concentration of orlistat was >14 µM for 14-03/F7 and >43 µM for WB-C6, respectively. Importantly, the combination of both drugs revealed no interaction on their inhibitory effects. We demonstrate that orlistat is a potent inhibitor of G. duodenalis growth in vitro and kills parasites at concentrations achievable in the gut by approved treatment regimens for obesity. We therefore propose to investigate orlistat in controlled clinical studies as a new drug in giardiasis.     

Size-Dependent Toxicity of Silver Nanoparticles to Bacteria,Yeast, Algae,Crustaceans and Mammalian Cells In Vitro

The concept of nanotechnologies is based on size-dependent properties of particles in the 1–100 nm range. However, the relation between the particle size and biological effects is still unclear. The aim of the current paper was to generate and analyse a homogenous set of experimental toxicity data on Ag nanoparticles (Ag NPs) of similar coating (citrate) but of 5 different primary sizes (10, 20, 40, 60 and 80 nm) to different types of organisms/cells commonly used in toxicity assays: bacterial, yeast and algal cells, crustaceans and mammalian cells in vitro. When possible, the assays were conducted in ultrapure water to minimise the effect of medium components on silver speciation. The toxic effects of NPs to different organisms varied about two orders of magnitude, being the lowest (∼0.1 mg Ag/L) for crustaceans and algae and the highest (∼26 mg Ag/L) for mammalian cells. To quantify the role of Ag ions in the toxicity of Ag NPs, we normalized the EC₅₀ values to Ag ions that dissolved from the NPs. The analysis showed that the toxicity of 20–80 nm Ag NPs could fully be explained by released Ag ions whereas 10 nm Ag NPs proved more toxic than predicted. Using E. coli Ag-biosensor, we demonstrated that 10 nm Ag NPs were more bioavailable to E. coli than silver salt (AgNO₃). Thus, one may infer that 10 nm Ag NPs had more efficient cell-particle contact resulting in higher intracellular bioavailability of silver than in case of bigger NPs. Although the latter conclusion is initially based on one test organism, it may lead to an explanation for “size-dependent“ biological effects of silver NPs. This study, for the first time, investigated the size-dependent toxic effects of a well-characterized library of Ag NPs to several microbial species, protozoans, algae, crustaceans and mammalian cells in vitro.     

JSI-124 Suppresses Invasion and Angiogenesis of Glioblastoma Cells In Vitro

Glioblastoma multiforme (GBM) is one of the utmost malignant tumors. Excessive angiogenesis and invasiveness are the major reasons for their uncontrolled growth and resistance toward conventional strategies resulting in poor prognosis. In this study, we found that low-dose JSI-124 reduced invasiveness and tumorigenicity of GBM cells. JSI-124 effectively inhibited VEGF expression in GBM cells. In a coculture study, JSI-124 completely prevented U87MG cell–mediated capillary formation of HUVECs and the migration of HUVECs when cultured alone or cocultured with U87MG cells. Furthermore, JSI-124 inhibited VEGF-induced cell proliferation, motility, invasion and the formation of capillary-like structures in HUVECs in a dose-dependent manner. JSI-124 suppressed VEGF-induced p-VEGFR2 activity through STAT3 signaling cascade in HUVECs. Immunohistochemistry analysis showed that the expression of CD34, Ki67, p-STAT3 and p-VEGFR2 protein in xenografts was remarkably decreased. Taken together, our findings provide the first evidence that JSI-124 effectively inhibits tumor angiogenesis and invasion, which might be a viable drug in anti-angiogenesis and anti-invasion therapies.     

Anti-Inflammatory Activity of N-Butanol Extract from Ipomoea stolonifera In Vivo and In Vitro

Ipomoea stolonifera (I. stolonifera) has been used for the treatment of inflammatory diseases including rheumatism and rheumatoid arthritis in Chinese traditional medicine. However, the anti-inflammatory activity of I. stolonifera has not been elucidated. For this reason, the anti-inflammatory activity of n-butanol extract of I. stolonifera (BE-IS) was evaluated in vivo by using acute models (croton oil-induced mouse ear edema, carrageenan-induced rat paw edema, and carrageenan-induced rat pleurisy) and chronic models (cotton pellet-induced rat granuloma, and complete Freund’s adjuvant (CFA)-induced rat arthritis). Results indicated that oral administration of BE-IS significantly attenuated croton oil-induced ear edema, decreased carrageenan-induced paw edema, reduced carrageenan-induced exudates and cellular migration, inhibited cotton pellet-induced granuloma formation and improved CFA-induced arthritis. Preliminary mechanism studies demonstrated that BE-IS decreased the levels of myeloperoxidase (MPO) and malondialdehyde (MDA), increased the activity of anti-oxidant enzyme superoxide dismutase (SOD) in vivo, and reduced the production of nitric oxide (NO), prostaglandin E₂ (PGE₂), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in lipopolysaccharide-activated RAW264.7 macrophages in vitro. Results obtained in vivo and in vitro demonstrate that BE-IS has considerable anti-inflammatory potential, which provided experimental evidences for the traditional application of Ipomoea stolonifera in inflammatory diseases.     

Triclabendazole Sulfoxide Causes Stage-Dependent Embryolethality in Zebrafish and Mouse In Vitro

Background

Fascioliasis and paragonimiasis are widespread foodborne trematode diseases, affecting millions of people in more than 75 countries. The treatment of choice for these parasitic diseases is based on triclabendazole, a benzimidazole derivative which has been suggested as a promising drug to treat pregnant women and children. However, at the moment, this drug is not approved for human use in most countries. Its potential adverse effects on embryonic development have been scarcely studied, and it has not been assigned a pregnancy category by the FDA. Thus, to help in the process of risk-benefit decision making upon triclabendazole treatment during pregnancy, a better characterization of its risks during gestation is needed.

Methodology

The zebrafish embryo test, a preimplantation and a postimplantation rodent whole embryo culture were used to investigate the potential embryotoxicity/teratogenicity of triclabendazole and its first metabolite triclabendazole sulfoxide. Albendazole and albendazole sulfoxide were included as positive controls.

Principal Findings

Triclabendazole was between 10 and 250 times less potent than albendazole in inducing dysmorphogenic effects in zebrafish or postimplantation rodent embryos, respectively. However, during the preimplantation period, both compounds, triclabendazole and triclabendazole sulfoxide, induced a dose-dependent embryolethal effect after only 24 h of exposure in rodent embryos and zebrafish (lowest observed adverse effect concentrations = 10 μM).

Conclusions/Significance

In humans, after ingestion of the recommended doses of triclabendazole to treat fascioliasis and paragonimiasis (10 mg/kg), the main compound found in plasma is triclabendazole sulfoxide (maximum concentration 38.6 μM), while triclabendazole concentrations are approximately 30 times lower (1.16 μM). From our results it can be concluded that triclabendazole, at concentrations of the same order of magnitude as the clinically relevant ones, does not entail teratogenic potential in vitro during the organogenesis period, but its first metabolite triclabendazole sulfoxide has a high embryotoxic capacity in vitro during the preimplantation stage.