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Co-cultures of entorhinal cortex (EC) and dentate gyrus (DG) explants are a useful model system to study the formation and stabilization of axonal projections. We adapted this model system to EC-DG co-cultures on microelectrode arrays (MEA) for the characterization of axonal projections on a functional level for days and weeks. EC and DG explants were placed on MEA to allow for the reconstitution of perforant pathway projections. Connections formed were characterized by morphological and electrophysiological analyses to verify characteristic features of perforant pathway signal transmission. Morphological analysis reveals proper projection of EC neurons into the molecular layer of the DG. Examination of synaptic transmission after high frequency stimulation imply unidirectional connections that used glutamate receptors of the AMPA/kainate type as main mediators of excitatory signal transmission. The system was evaluated by the introduction of the NCAM binding peptide C3d. In accordance with in vivo and in vitro experiments C3d modulated signal transmission by NCAM-related mechanisms resulting in morphological re-arrangements.

The analysis of parameters in bronchoalveolar extracellular lining secretions has come into greater use in the diagnosis of diseases of the lung and respiratory passages. The bronchoalveolar lavage (BAL) method is thus used for sampling alveolar fluids or bronchial secretions. However, this method is invasive and therefore cannot be routinely employed for probe sampling. Based on the hypothesis that aerosol particles excreted in human breath reflect the composition of the bronchoalveolar extracellular lining fluid, experiments were performed to concentrate and analyze these aerosols directly using a noninvasive technique. Human exhaled air was directed through a set of cool traps and the condensate of 200 to 400 exhalations examined for nonvolatile components, such as proteins. In experiments conducted with volunteers, the amount of proteins in the breath condensate of 8 healthy individuals (of a total of 10) amounted to between 4 micrograms and 1.4 mg. The proteins were separated by two-dimensional polyacrylamide gel electrophoresis (PAGE) and compared to saliva samples of the respective volunteers. The results suggest that the proteins detected in breath originate partially from the naso-oropharyngeal tract and partially from lower regions of the airways. In clinical tests, the exhaled air of 13 patients suffering from various diseases of the respiratory tract was sampled and analyzed by immunoassays for inflammation parameters, such as interleukin-1 beta (IL-1 beta), soluble interleukin-2 receptor protein, light chain (sIL-2R), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha). In these tests, up to 370 pg IL-1 beta, 120 pg TNF-alpha, and 2,159 U sIL-2R per ml were measured in the breath condensate.(ABSTRACT TRUNCATED AT 250 WORDS)

Field potential analysis can supplement network studies with information on graded neuronal activity in large populations and the physiological state of the tissue. In extracellular recordings with a microelectrode-array the authors investigated the structure of local field potentials (ERG) induced by visual stimulation and their temporal relation to spike activity in excised chicken retina segments. To assess the influence of tissue viability on both parameters the authors evaluated spike response characteristics and ERG after prolonged recording and spreading depression, a pathological phenomenon in neural tissue. Spike characteristics corresponded well to in vivo recordings with mainly phasic ON- and OFF-responses. ERGs consisted of four components differentially and reproducibly varying with the physiological state of the tissue.

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.

In order to quantify autoantibodies in the sera of patients with autoimmune disease, we have created a microarray-based immunoassay that allows the simultaneous analysis of 18 known autoantigens. The microarrays contain serial dilutions of the various antigens, thereby allowing accurate determination of autoantibody titer using minimal amounts of serum. The assay is very sensitive and highly specific: as little as 40 fg of a known protein standard can be detected with little or no cross-reactivity to nonspecific proteins. The signal intensities observed from serial dilutions of immobilized antigen correlate well with serial dilutions of autoimmune sera. Miniaturized and highly parallelized immunoassays like these will reduce costs by decreasing reagent consumption and improve efficiency by greatly increasing the number of assays that can be performed with a single serum sample. This system will significantly facilitate and accelerate the diagnostics of autoimmune diseases and can be adapted easily to any other kind of immunoassay.

Analysis of developmental mechanisms during neuroembryogenesis, evaluation of toxicological effects and testing of neuroprotheses rely to an increasing extent on in vivo-like in vitro models. We have developed a novel organotypic culture system of the chick retina. Tissue slices of embryonic retinae were immobilized on glass coverslips by a fibrin clot and permanently rotated between the gas and medium phase, resulting in regular formation and the maintenance of the retinal cytoarchitecture. Selection of embryonic stage, slice thickness and specimen processing were optimized for culturing. Scanning electron microscopy revealed degradation during increasing culture periods of the fibrin clot, which was used for initial immobilization of explants on glass coverslips. Simultaneously, retinal cells became exposed on the tissue surface. Even after several weeks in vitro, formation and maintenance of plexiform and nuclear layers was evident as revealed by two specific monoclonal antibodies. Immunocytochemistry employing two additional photoreceptor- and radial Muller-antibodies indicated differentiation of neuronal and glial cells specific for the retina. The organotypic culture system promises to facilitate developmental studies of retinal development. Quantitative evaluation of Na + -channel blocker mexiletine impact on the histogenesis of retinal explants proved the organotypic culture system to be a valuable tool also for neurotoxicological investigations. Themes: Theme A Topics: topic: 20.1

Experiments were performed to analyze whether short-term exposure to a sinusoidal extremely low-frequency electromagnetic field (20 Hz, 8 mT) can alter the dynamics of intracellular calcium in diploid human skin fibroblasts. In heterogeneous fibroblast populations, about 30% of the cells responded with a change in the oscillation activity of intracellular calcium within 40 min. It was demonstrated at the level of the single cell that the responsiveness of fibroblast populations to extremely low-frequency electromagnetic fields depends on the specific differentiation state of the exposed cell. The data obtained clearly indicate that mitotic progenitor fibroblasts respond with an enhancement of the dynamics of calcium, whereas in postmitotic fibrocytes a reduction of the dynamics was observed when the cells were co-stimulated with suboptimal concentrations of platelet-derived growth factor. Thus data from our laboratory on terminal differentiation induced by extremely low-frequency electromagnetic fields may be correlated with changes in the dynamics of Ca2+ reported here.

Stationary and dynamic fluorescence experiments with pyrene as a probe molecule indicate higher aggregation numbers in Triton-X-100 micelles at higher temperatures well below the cloud point. The structure of the micelles is changed considerably as (in spite of higher aggregation) the hydrophobic compartment available to the probe molecules becomes smaller. The increase of aggregation is combined with a decrease of the number of micelles, i. e. micelles are dissolved or merge with others.

The goal of this study is to quantify smooth muscle myosin (SMM) expression at the level of the individual cell and to ascertain whether SMM expression in cultured aortic smooth muscle cells is related to definite growth phases, and whether the initial seeding density affects growth or SMM staining. Rabbit aortic smooth muscle cells (SMCs) were harvested by enzyme digestion of aortic tissue and plated at low (100 cells cm-2), medium (1000 cells cm-2), and high (10,000 cells cm-2) densities. Independent of seeding density, the lag phase lasted 2 to 3 days and, at all three densities, the growth rate during the logarithmic growth phase was almost the same. However, the time, the number of population doubling needed to reach the plateau phase and the cell number in the plateau were influenced by the initial seeding density. Immunofluorescence staining with anti-smooth muscle myosin (ASMM) revealed intensive staining of striated and filamentous patterns in all cells during the lag and early logarithmic growth phases. During the late logarithmic growth phase, two subpopulations of cells appeared, one showing a positive and the other no reaction with SMM antiserum. The lowest relative number of cells which showed positive reactions with SMM antiserum was observed toward the end of the logarithmic growth phase. During the plateau phase, the SMM-positive subpopulation increased, amounting to about 60% of the total number of cells, independent of the seeding density. In terms of absolute numbers, the number of SMM-positive cells increased over the course of 21 days by factors of 13, 72, and 342 for high, medium, and low seeded cultures, respectively. We conclude that a SMC subpopulation can divide without loss of SMM and that some, but not all, cells which lose their SMM may possibly regain it in the postconfluent state.

The time course of structural changes in vascular smooth muscle cells (SMC) was investigated during the formation of an experimental lesion in response to balloon injury. We compared the filamentous organization, evaluated by quantitative electron microscopy, with the cellular content of two representative cytocontractile proteins (myosin and tropomyosin) as assessed by immunofluorescence. We found that the changes peak between 7 and 14 days after injury and that they are visible both in the neointima and to a lesser extent in the inner media. While virtually all SMC are of a filament-rich phenotype in the undisturbed media, after balloon injury SMC migrated into the intima and about 90% of these latter cells were either of a organelle-rich or an intermediate phenotype, with the remaining 10% being of the filament-rich phenotype. In the inner media about 40% of cells were either of organelle-rich or intermediate phenotype. In contrast to these profound organizational changes of responding SMC, histochemistry revealed only a slight and probably transient decrease of the cellular content of myosin and tropomyosin at that time point. Twenty-eight days after injury the discrepancies between the content and the organization of cytocontractile proteins became more apparent. While virtually all SMC showed a homogeneous intensive staining with both antibodies, indistinguishable from the media SMC, the organization of cytoplasmic filaments had not totally recovered. Even though this morphological study does not permit conclusions to be drawn on the contractile function of the cells, it shows that both the organization and the content of cytocontractile proteins have to be analyzed and compared for SMC changes to be evaluated during the formation of an experimental lesion.

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.