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Electroosmotic perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures

Artikel i vetenskaplig tidskrift
Författare Y. G. Ou
J. F. Wu
Mats Sandberg
S. G. Weber
Publicerad i Analytical and Bioanalytical Chemistry
Volym 406
Nummer/häfte 26
Sidor 6455-6468
ISSN 1618-2642
Publiceringsår 2014
Publicerad vid Institutionen för biomedicin, avdelningen för medicinsk kemi och cellbiologi
Sidor 6455-6468
Språk en
Länkar dx.doi.org/10.1007/s00216-014-8067-...
Ämnesord Organotypic hippocampal slice cultures, membrane-bound enzymes, enzyme activity, electroosmotic, PUSH-PULL PERFUSION, HORMONE-DEGRADING ECTOENZYME, CONVECTION-ENHANCED, DELIVERY, ELECTROLYTE-FILLED CAPILLARY, SINGLE-CELL ELECTROPORATION, IN-VIVO, COENZYME-A, ELECTROCHEMICAL DETECTION, AMINOPEPTIDASE-N, BRAIN-TISSUE, Biochemical Research Methods, Chemistry, Analytical
Ämneskategorier Cellbiologi, Biokemi

Sammanfattning

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 similar to 100 mu m spatial resolution, which is important for studies of enzyme kinetics in adjacent regions of the rat hippocampus.

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