A neural extracellular matrix-based method for in vitro hippocampal neuron culture and dopaminergic differentiation of neural stem cells

Background: The ability to recreate an optimal cellular microenvironment is critical to understand neuronal behavior and functionality in vitro. An organized neural extracellular matrix (nECM) promotes neural cell adhesion, proliferation and differentiation. Here, we expanded previous observations on the ability of nECM to support in vitro neuronal differentiation, with the following goals: (i) to recreate complex neuronal networks of embryonic rat hippocampal cells, and (ii) to achieve improved levels of dopaminergic differentiation of subventricular zone (SVZ) neural progenitor cells. Methods: Hippocampal cells from E18 rat embryos were seeded on PLL- and nECM-coated substrates. Neurosphere cultures were prepared from the SVZ of P4-P7 rat pups, and differentiation of neurospheres assayed on PLL- and nECM-coated substrates. Results: When seeded on nECM-coated substrates, both hippocampal cells and SVZ progenitor cells showed neural expression patterns that were similar to their poly-L-lysine-seeded counterparts. However, nECM-based cultures of both hippocampal neurons and SVZ progenitor cells could be maintained for longer times as compared to poly-L-lysine-based cultures. As a result, nECM-based cultures gave rise to a more branched neurite arborization of hippocampal neurons. Interestingly, the prolonged differentiation time of SVZ progenitor cells in nECM allowed us to obtain a purer population of dopaminergic neurons. Conclusions: We conclude that nECM-based coating is an efficient substrate to culture neural cells at different stages of differentiation. In addition, neural ECM-coated substrates increased neuronal survival and neuronal differentiation efficiency as compared to cationic polymers such as poly-L-lysine.

Mantenimiento en el laboratorio del pez cebra

El pez cebra (Danio rerio) se utiliza como organismo modelo en distintos campos como la biomedicina o la toxicogenómica. Las ventajas que ofrece frente a otros modelos animales, hace que se haya convertido en los últimos años en uno de los organismos modelo más utilizado en experimentación. La similitud de su desarrollo embrionario con el de otros vertebrados superiores o la semejanza de su genoma con la del ser humano lo han colocado en el punto de mira de las principales investigaciones. Pero disponer de ejemplares óptimos tanto de embriones como de adultos para su utilización en investigación, requiere de unos cuidados y de una atención adecuada. Para su mantenimiento se deberán tener en cuenta el tipo de instalaciones para su cría, la calidad del agua donde se encuentran, la alimentación que reciben o los procesos de reproducción utilizados. El objetivo de este estudio ha sido elaborar un protocolo de mantenimiento del pez cebra, que optimice los cuidados necesarios para que el número de embriones viables sea máximo y también poder mantener un número de ejemplares adultos en buenas condiciones.

Evaluation of marine phytoplankton toxicity by application of marine invertebrate bioassays

The dinoflagellate Alexandrium minutum and the haptophyte Prymnesium parvum are well known for their toxin production and negative effects in marine coastal environments. A. minutum produces toxins which cause paralytic shellfish poisoning in humans and can affect copepods, shellfish and other marine organisms. Toxins of P. parvum are associated with massive fish mortalities resulting in negative impacts on the marine ecosystem and large economic losses in commercial aquaculture. The aim of this work is to improve our knowledge about the reliability of the use of marine invertebrate bioassays to detect microalgae toxicity, by performing: (i) a 24- to 48-h test with the brine shrimp Artemia franciscana; (ii) a 48-hour embryo-larval toxicity test with the sea urchin Paracentrotus lividus; and (iii) a 72-h test with the amphipod Corophium multisetosum. The results indicate that A. franciscana and P. lividus larvae are sensitive to the toxicity of A. minutum and P. parvum. LC50 comparison analysis between the tested organisms reveals that A. franciscana is the most sensitive organism for A. minutum. These findings suggest that the use of different organizational biological level bioassays appears to be a suitable tool for A. minutum and P. parvum toxicity assessment.

Mechanisms of Toxicity of Ag Nanoparticles in Comparison to Bulk and Ionic Ag on Mussel Hemocytes and Gill Cells

Silver nanoparticles (Ag NPs) are increasingly used in many products and are expected to end up in the aquatic environment. Mussels have been proposed as marine model species to evaluate NP toxicity in vitro. The objective of this work was to assess the mechanisms of toxicity of Ag NPs on mussel hemocytes and gill cells, in comparison to ionic and bulk Ag. Firstly, cytotoxicity of commercial and maltose stabilized Ag NPs was screened in parallel with the ionic and bulk forms at a wide range of concentrations in isolated mussel cells using cell viability assays. Toxicity of maltose alone was also tested. LC50 values were calculated and the most toxic Ag NPs tested were selected for a second step where sublethal concentrations of each Ag form were tested using a wide array of mechanistic tests in both cell types. Maltose-stabilized Ag NPs showed size-dependent cytotoxicity, smaller (20 nm) NPs being more toxic than larger (40 and 100 nm) NPs. Maltose alone provoked minor effects on cell viability. Ionic Ag was the most cytotoxic Ag form tested whereas bulk Ag showed similar cytotoxicity to the commercial Ag NPs. Main mechanisms of action of Ag NPs involved oxidative stress and genotoxicity in the two cell types, activation of lysosomal AcP activity, disruption of actin cytoskeleton and stimulation of phagocytosis in hemocytes and increase of MXR transport activity and inhibition of Na-K-ATPase in gill cells. Similar effects were observed after exposure to ionic and bulk Ag in the two cell types, although generally effects were more marked for the ionic form. In conclusion, results suggest that most observed responses were due at least in part to dissolved Ag.