Novel microdialysis method to assess neuropeptides and large molecules in free-moving mouse.
ABSTRACT Microdialysis is a powerful in vivo technique for the continuous sampling of small molecules within the extracellular fluid space. However, efforts to collect larger molecules have met with little success. To identify biologically active larger molecules in free-moving animals would be of great benefit. For this purpose, we have developed a novel microdialysis method that allows consistent recovery of large molecules from the brain interstitial space in the awake, free-moving mouse. Using a new "vent" probe with a push-pull perfusion system, the present study successfully demonstrated in vivo sampling of pathophysiologically important macromolecules in free-moving mouse brain. This sampling system allowed monitoring of the dynamic changes in their concentrations. Overall, this novel microdialysis system would provide the opportunity to identify the expression patterns of pathophysiologically important proteins in a variety of physiological and pathological processes for a better understanding of various diseases.
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ABSTRACT: This review covers recent advances in sampling fluid from the extracellular space of brain tissue by electroosmosis (EO). Two techniques, EO sampling with a single fused-silica capillary and EO push-pull perfusion, have been developed. These tools were used to investigate the function of membrane-bound enzymes with outward-facing active sites, or ectoenzymes, in modulating the activity of the neuropeptides leu-enkephalin and galanin in organotypic-hippocampal-slice cultures (OHSCs). In addition, the approach was used to determine the endogenous concentration of a thiol, cysteamine, in OHSCs. We have also investigated the degradation of coenzyme A in the extracellular space. The approach provides information on ectoenzyme activity, including Michaelis constants, in tissue, which, as far as we are aware, has not been done before. On the basis of computational evidence, EO push-pull perfusion can distinguish ectoenzyme activity with a ~100 μm spatial resolution, which is important for studies of enzyme kinetics in adjacent regions of the rat hippocampus.Analytical and Bioanalytical Chemistry 08/2014; 406(26). DOI:10.1007/s00216-014-8067-2 · 3.58 Impact Factor
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 01/2015; 35(2):518-526. DOI:10.1523/JNEUROSCI.3742-14.2015 · 6.75 Impact Factor
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ABSTRACT: The purpose of this study was to evaluate the therapeutic potential of human induced pluripotent stem (iPS) cell-derived macrophage-like cells for Alzheimer’s disease (AD). In previous studies, we established the technology to generate macrophage-like myeloid lineage cells with proliferating capacity from human iPS cells, and we designated the cells iPS-ML. iPS-ML reduced the level of Aβ added into the culture medium, and the culture supernatant of iPS-ML alleviated the neurotoxicity of Aβ. We generated iPS-ML expressing the Fc-receptor-fused form of a single chain antibody specific to Aβ. In addition, we made iPS-ML expressing Neprilysin-2 (NEP2), which is a protease with Aβ-degrading activity. In vitro, expression of NEP2 but not anti-Aβ scFv enhanced the effect to reduce the level of soluble Aβ oligomer in the culture medium and to alleviate the neurotoxicity of Aβ. To analyze the effect of iPS-ML expressing NEP2 (iPS-ML/NEP2) in vivo, we intracerebrally administered the iPS-ML/NEP2 to 5XFAD mice, which is a mouse model of AD. We observed significant reduction in the level of Aβ in the brain interstitial fluid following administration of iPS-ML/NEP2. These results suggested that iPS-ML/NEP2 may be a potential therapeutic agent in the treatment of AD.Stem Cell Research 10/2014; 13(3). DOI:10.1016/j.scr.2014.10.001 · 3.91 Impact Factor