In vitro neurotoxicology tests based on the coupling of brain slices to silicon microelectrode arrays

Leistungen

The technological developments carried out allowed the fabrication of microelectrode arrays (MEAs) consisting of planar and two types of 3-dimensional Pt electrodes integrated on planar or perforated Si/silicon nitride substrate. In addition, the technology for the realisation of the planar and hillock-type MEAs on glass substrates has been developed. These constitute an alternative if transparent devices are required. Continuously upgraded during the project, the final generation of the MEAs forms a catalogue of devices for different cell cultures (acute, organotypic) and electrophysiological experiments. More than 200 devices of different types have been provided and their evaluation carried out by the project partners. Constraints arising from technological and biological issues have been taken into consideration and several aspects addressed during the development for optimising the MEAs particularly in terms of cell culture survival, electrical performance (electrode/cell coupling) and convenience of use (robustness, number of recording sites, substrate porosity). The outcome of this development are altogether six generations of MEAs integrating planar and/or 3-dimensional Pt microelectrodes on a planar or a perforated silicon/silicon nitride substrate. The main technological developments have been carried out in the areas of the electroplating of bright Pt, perforation by DRIE of the Si/Si3N4 substrate and Pt-tip realisation by the anisotropic etching. The MEAs consist of 32 Pt microelectrodes and four reference electrodes ordered in a rectangle and embedded on a Si/Si3N4 substrate. Three types of geometries of Pt microelectrodes namely planar, hillock-type and tip have been fabricated. The technologies used were thin-film deposition, electroplating and Si anisotropic etching/thin-film deposition respectively.

It has been demonstrated that slices of developing brain tissue (e. g. rat hippocampus) can be grown for extended periods of time (weeks) on an silicon-based microelectrode array, which was manufactured for the purpose of multiple site, long term electrophysical recordings (and stimuations) of neurons and neuronal connections (during development and after maturation) and for sensitive and early detection of functional neurotoxic effects of know and novel compounds in comparison with established biomarkers for structural and neurodegenerative changes. The results were obtained in parallel with studies of slice cultures grown by conventional techniques on semiporous membranes, which also served to establish standardized protocols for reproducible exposure paradigms and quantitative image analysis with monitoring of induced neuronal cell death and structural changes. We have shown that brain slices growing on the array can be exposed to chemicals and that the electrical neuronal activity can be measured by connecting the electrodes of the array to conventional electrophysiological recording equipment. The brain tissue can be stimulated electrically through the array or chemically by adding chemicals to the culture medium.The method can be used to study acute and long-term physiological actions of drugs, toxins and other biologically active substances on cultured, organotypically organized brain slices, as well as for on-line and long-term studies of developmental and regenerative processes in brain tissues. The result includes proof of biocompatibility and long-term support of developing brain tissue slices and hence the basic feasiability of the silicon-based microelectrode array, manufactured by Neuchatel-IMT. Included is also the demonstration by Odense-IMB of intimate structural contact between the uninsulated tips of the microelectrodes and neuronal processes in the cultured brain slices, which comply with the electrophysiological results (sensitivity and signal to noise ratios), obtained by Copenhagen-DMP. The result combines the fields of neurobiology/cell biology (A14), toxicology (A38),microelectronics (D23) and manufacturing technology. The culturing of brain tissue slices for extended periods of time on microelectrode arrays with pointed electrodes penetrating deep into and firmly positioned inside the cultured slice is unique, and must in terms of qualities of electrophysiological recordings be considered superior to the growth of brain slices on devices with planar or minor pointed electrodes available on the market. The result includes a series of procedures some of which are modifications of existing and published procedures for obtaining and maintenance of tissue for organotypic slice cultures of the mammalian brain. Using the procedures it is possible to harvest sections of brain from newborn (to 2 week old) rats and place the sections in an incubator for maturation. After about one week the organotypic culture is ready to be placed on a microelectrode array and be maintained on the array for up to a week at present. During this period the tissue on the array can be transferred to a set-up and connected to standard electrophysiological equipment. Thus the physiology of the tissue and the effect of drugs and chemicals including toxins can be studied in vitro for several days with the existing prototype and protocol.

We developed a protocol for in vitro testing of neurotoxicity in freshly cut brain slices. Within the scientific and industrial community the need for better, novel tests of in vitro neurotoxicity is recognized. Electrophysiology is a novel, little explored source of neurotoxicity endpoints. Electrophysiological endpoints are easy to detect and very sensitive to toxicity. In fact, they relate directly to the functioning of neural cells, not just to morphological of biochemical features which somehow relate to toxicity. The protocol we developed proved capable to detect within 1 hour toxicity by N-methyl-d-aspartate (NMDA), kainic acid (KA) and tri-methyl-tin (TMT). Within this time span it was superior to a histological test (MAP2 density measurement) in detecting toxicity. Moreover, it proved capable to differentiate epileptogenic toxins (NMDA, KA) from a weakly excitatory one (not epileptogenic) like TMT, based on the analysis of bursting responses to single stimuli. Additionally, the same protocol was carried out for use with the 3-dimensional microelectrode array that our consortium developed. Using the latter, it was also carried out in slices of human brain tissue taken at the time of neurosurgery. Under all conditions the protocol proved reliable in detecting toxicity by kainic acid.

Leistungen

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