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

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.

Numerical Modeling of Electroosmotic Push–Pull Perfusion and Assessment of Its Application to Quantitative Determination of Enzymatic Activity in the Extracellular Space of Mammalian Tissue

“…However, the ζ-potential of fused silica capillaries, −46 mV 17 , is different from that of the tissue, −23 mV 41 . For an incompressible fluid in a closed system, EO flow through a medium with non-uniform ζ-potentials results in non-uniform EO velocities.…”

“…This last section addresses how to obtain quantitative information about enzyme activity in the tissue from the perfusion data. An example reaction that can occur in the tissue is as follows: $$\mathrm{normalYGGFL}\to \mathrm{normalY}+\mathrm{normalGGFL}$$where YGGFL is Leu-enkephalin, a common endogenous opioid peptide in the brain ECS, and GGFL is the major hydrolysis product after cleavage by membrane-bound aminopeptidase(s) whose catalytic domains face the ECS 17,28,33 . The following discussion assumes a Michaelis-Menten model but other appropriate models could also be used.…”

Temperature-based on-column solute focusing in capillary liquid chromatography reduces peak broadening from pre-column dispersion and volume overload when used alone or with solvent-based focusing

“…Over a 5-minute sampling time, we collect 50 – 125 nL of the perfusion solution containing peptide and hydrolysis products. This volume is diluted to 10 or 15 μL depending on the application [28–30]. The final concentration of galanin in the injected samples is a few micromolar.…”

Methods of Measuring Enzyme Activity Ex Vivo and In Vivo

“…Electroosmosis (EO) is the bulk fluid movement that occurs when an electric current is passed through electrolyte-filled conduits with charged walls, e.g. fused-silica capillaries (98) and the brain ECS (99). There are two generations of EO-based sampling techniques for measuring enzyme activity ex vivo in tissue cultures, as described below.…”

“…Membrane-bound enzymes whose catalytic domains face the ECS can hydrolyze substrates carried by EO flow. The activity of these enzymes, called ectopeptidases, has been of great interest in recent years when it was discovered that they regulate the bioactivity of important peptides involved in growth, cell survival, and stress responses (reviewed in Ou et al (98)). Several such as dipeptidylpeptidase IV, and neutral endopeptidase have also become important therapeutic targets (101–103).…”