Wilfried Nisch

The Development of Subretinal Microphotodiodes for Replacement of Degenerated Photoreceptors

There are presently several concepts to restore vision in blind or highly visually handicapped persons by implanting electronic devices into the eye in order to partially restore vision. Here, the approach to replace retinal photoreceptors by a subretinally implanted microphotodiode array (MPDA) is summarized. A survey is given on the present state of the development of MPDAs, the possibility of in vitro and in vivo tests as well as first results on biocompatibility and histology. Additionally, electrophysiological recordings in rabbits and rats are presented which have received such subretinal implants.

A novel organotypic long-term culture of the rat hippocampus on substrate-integrated multielectrode arrays.

Spatiotemporally coordinated activity of neural networks is crucial for brain functioning. To understand the basis of physiological information processing and pathological states, simultaneous multisite long-term recording is a prerequisite. In a multidisciplinary approach we developed a novel system of organotypically cultured rat hippocampal slices on a planar 60-microelectrode array (MEA). This biohybrid system allowed cultivation for 4 weeks. Methods known from semiconductor production were employed to fabricate and characterize the MEA. Simultaneous extracellular recording of local field potentials (LFPs) and spike activity at 60 sites under sterile conditions allowed the analysis of network activity with high spatiotemporal resolution. To our knowledge this is the first realization of hippocampus cultured organotypically on multi-microelectrode arrays for simultaneous recording and electrical stimulation. This biohybrid system promises to become a powerful tool for drug discovery and for the analysis of neural networks, of synaptic plasticity, and of pathophysiological conditions such as ischemia and epilepsy.

Biostability of micro-photodiode arrays for subretinal implantation

Micro-photodiode arrays based on semiconductor chip technology are being developed to replace degenerated photoreceptor cells in the retina. Electric current is generated in tiny micro-photodiodes and delivered to the adjacent tissue by micro-electrodes. One of the main requirements of a sub-retinal implantable device is long-term stability versus corrosion in vivo (biostability). Biostability of micro-photodiode arrays (MPDA) was investigated in vitro and in vivo. No significant damage was found on chips immersed for up to 21 months in saline solution. Under in vivo conditions, however, the silicon oxide passivation layer of the chip was dissolved within a period of about 6-12 months. Subsequently, the underlying silicon was corroded. In contrast, stimulation electrodes consisting of titanium nitride were well preserved both in vitro and in vivo. The deterioration of the electrical properties of the micro-photodiodes correlated with the morphological damage observed. Strategies aiming at the development of an improved biostable encapsulation of neurotechnological implants have to be investigated and will be discussed briefly.

A thin film microelectrode array for monitoring extracellular neuronal activity in vitro.

A planar array of microelectrodes has been developed for monitoring the electrical activity of neurons in cell culture. The microelectrode array was tested and characterized using impedance measurements and SEM. To verify the spatial sensitivity of the microelectrodes we used a specially developed simulation device.

Novel thin film titanium nitride micro-electrodes with excellent charge transfer capability for cell stimulation and sensing applications

Microelectrodes with high specific charge transfer capability are an important prerequisite for high resolution stimulation, and recording of neuronal and muscular tissue. Novel thin film titanium nitride (TiN) microelectrodes were fabricated onto a microelectrode array by reactive sputtering in a nitrogen/argon atmosphere. The electrodes showed excellent charge transfer capacity of 40 mC/cm/sup 2/ and low ac-impedance along with high pulse stability. Scanning electron microscopy revealed a regular columnar morphology providing for a large internal surface area. With respect to their electrical and mechanical properties the TiN electrodes proved superior to electrodes produced by anodic oxidation of iridium. In this type of iridium oxide irregular cracks formed probably due to the high density ratio between metal and oxide. Furthermore, in spite of high charge capacity at very low frequencies (Q/sub cap/) the iridium oxide electrodes exhibited poor safe charge injection limit under pulsing conditions (Q/sub inj/).

Extracellular recording in neuronal networks with substrate integrated microelectrode arrays

A photolithographically produced array of 60 substrate-integrated microelectrodes was used for extracellular recording. Neuronal electrical activity was recorded from chicken retinal ganglion cells with or without stimulation by diffuse light. The retina was removed from chicken embryos of embryonic day 14-18. Only cells recorded from day 18 retina would react to photostimulation, increasing their activity when stimulated, corresponding to the developmental time course of photoreceptor differentiation.

Patch-clamping of primary cardiac cells with micro-openings in polyimide films

Patch-clamping is a powerful method for investigating the function and regulation of ionic channels. Currently, great efforts are being made to automate this method. As a step towards this goal, the feasibility of patch-clamping primary cells with a microscopic opening in a planar substrate was tested. Using standard microfabrication and ion beam technology, small-diameter openings (2 and 4 μm) were formed in polyimide films (thickness 6.5 μm). Single cells (sheep Purkinje heart cells, Chinese hamster ovary cells) in a suspension were positioned on top of the opening and sucked towards the opening to improve adhesion of the cell to the planar substrate, hence increasing the seal resistance. Voltage/current measurements yielded a median seal resistance of 1.3MΩ with 4 μm openings (n=24) and 26.0 MΩ with 2 μm openings (n=75), respectively. With 2 μm openings, successful loose-patch recordings of TTX-sensitive inward currents and action potentials in sheep Purkinje heart cells (n=18) were made. In rare cases, gigaseals (n=4) were also measured, and a whole-cell configuration (n=1) could be established. It was concluded that the simple planar patch approach is suitable for automated loosepatch recordings from cells in suspension but will hardly be suitable for highthroughput whole-cell patch-clamping with high-resistance seals.

Subretinales Mikrophotodioden-Array als Ersatz für degenerierte Photorezeptoren?

ZusammenfassungEs wird eine Übersicht über den Stand der Entwicklungen eines subretinalen, elektronischen Mikrophotodioden-Arrays gegeben, mit dem degenerierte Photorezeptoren ersetzt werden können. Verschiedene Prototypen wurden entwickelt, getestet und bei verschiedenen Versuchstieren bis zur Dauer von 18 Monaten implantiert. Die Tatsache, dass mit subretinalen Elektroden elektrische Antworten vom visuellen Kortex von Schweinen abgeleitet werden können und Antworten auch in vitro in Netzhäuten von Ratten mit Netzhautdegenerationen nachweisbar sind, zeigt die Sinnhaftigkeit dieses Ansatzes. Allerdings sind noch wichtige Fragen der Biokompatibilität, der Langzeitstabilität und der Art des vermittelbaren Bildeindrucks zu bearbeiten, bevor an einen Einsatz beim Menschen zu denken ist.AbstractA survey is given on the status of developments, concerning a subretinal electronic microphotodiode array that aims at replacing degenerated photoreceptors. Various prototypes have been developed, tested, and implanted in various experimental animals up to 18 months. The fact that electrical responses were recorded from the visual cortex of pigs after electrical stimulation by subretinal electrodes and the fact that responses are also recorded in-vitro in degenerated rat retinae, shows the feasibility of this approach. However, there are a number of open questions concerning the biocompatibility, the long-time stability, and the type of transmitted image to be solved before application in patients can be considered.

Electrical properties of a light-addressable microelectrode chip with high electrode density for extracellular stimulation and recording of excitable cells

A light-addressable microelectrode chip with 3600 TiN electrodes was fabricated. Amorphous silicon (a-Si:H) serves as a photo conductor. The electrodes on the chip are addressed by a laser spot and electrical properties of the system are determined. DC measurements show a dark to bright dynamic of 10(6)-10(7). The AC impedance dynamic @ 1 kHz/100 mV and thus the signal-to-noise-ratio is determined to 60. This value is quite sufficient for electrophysiological measurements. For the first time, recordings from cardiac myocytes are reported using the principle of light-addressing. Measurements were done with a standard laser scan microscope (Zeiss LSM 410).

Low-impedance thin-film polycrystalline silicon microelectrodes for extracellular stimulation and recording

Abstract Polycrystalline silicon thin films were explored with respect to their application as low-impedance microelectrodes for extracellular stimulation and recording of cells. Microelectrode arrays (MEAs) comprising polysilicon microelectrodes were fabricated using CMOS-compatible processes. Overall capacitance of an electrode with a diameter of 20 μm is on the order of 200–300 pF. Chemical and morphological stability in physiological saline solution was excellent over a period of at least 5 months. This finding renders applications in neuronal implants or bio-chips. Nanoporous polysilicon electrodes were created by anodic oxidation in hydrofluoric acid (HF). However, no considerable decrease of electrode impedance was observed although pore formation was clearly confirmed by transmission electron microscopy (TEM).