Determination of ζ-Potential and Tortuosity in Rat Organotypic Hippocampal Cultures from Electroosmotic Velocity Measurements under Feedback Control

Abstract

Extracellular translational motion in the brain is generally considered to be governed by diffusion and tortuosity. However, the brain as a whole has a significant ζ-potential, thus translational motion is also governed by electrokinetic effects under a naturally occurring or applied electric field. We have previously measured ζ-potential and tortuosity in intact brain tissue, however the method was tedious. In this work, we use a four-electrode potentiostat to control the potential difference between two microreference electrodes in the tissue, creating a constant electric field. Additionally, some alterations have been made simplify our previous procedure. The method entails simultaneously injecting two 70 kDa dextran conjugated fluorophores into rat organotypic hippocampal cultures and observing their mobility using fluorescence microscopy. We further present two methods of data analysis: regression and two-probe analysis. Statistical comparisons are made between the previous and current methods as well as between the two data analysis methods. In comparison to the previous method, the current, simpler method with data analysis by regression gives statistically indistinguishable mean values of ζ-potential and tortuosity, with a similar variability for ζ-potential, −21.3 ± 2.8 mV, and a larger variability for the tortuosity, 1.98 ± 0.12. On the other hand, we find that the current method combined with the two-probe analysis produces accurate and more precise results, with a ζ-potential of −22.8 ± 0.8 mV and a tortuosity of 2.24 ± 0.10.Translational motion of molecules in the extracellular space of functioning tissues is typically viewed as being governed by diffusion and tortuosity. 1 Although not as well studied, electroosmotic effects occur in tissues due to natural processes 2 or experimentally applied fields. [3][4][5][6][7][8][9] Electroosmotic flow is the bulk fluid flow created by an electric field in a heterogeneous medium with a non-zero ζ-potential. In the brain for example, fixed charges on cell-surface functional groups and constituents of the extracellular matrix create a ζ-potential. 10 The electroosmotic velocity is governed by the magnitude of the ζ-potential.In order to determine the electroosmotic velocity in brain tissue, it is necessary to know the ζ-potential. Some methods exist for determining the ζ-potentials of particulate objects (e.g., cells) and film-like objects (e.g., skin). ζ-potentials of particulate objects have been determined by electrophoretic techniques. [11][12][13][14][15][16][17] Electroosmotic flow in thin samples, such as plant tissue can be determined by flux through the tissue. 18 Electroosmotic flow is described and quantitated as an influential parameter in transdermal iontophoresis [19][20][21][22][23][24][25] and transdermal sampling (reverse iontophoresis 24,26 applying an oscillating electric potential 27 or by scanning electrochemical microscopy. 25 The experimental arrangement for determining the electroosmotic velocity through pores, such as in skin ...

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“…[76] Finally, the brain itself, as a whole, has a specific ζ potential, which implies that the extracellular translational motion is governed by electrokinetic effects under this naturally occurring electric field in addition to diffusion and tissue tortuosity. [77] …”

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“…We have found that electroosmosis occurs in brain tissue [71, 72]. Electroosmosis is the result of the presence of an excess of mobile counterions near fixed surface charges.…”

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