| 1. |
- Guy, Yifat, et al.
(författare)
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Determination of zeta-potential and tortuosity in rat organotypic hippocampal cultures from electroosmotic velocity measurements under feedback control.
- 2009
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Ingår i: Analytical chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 81:8, s. 3001-7
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Tidskriftsartikel (refereegranskat)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 zeta-potential, thus translational motion is also governed by electrokinetic effects under a naturally occurring or applied electric field. We have previously measured zeta-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 to 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 zeta-potential and tortuosity, with a similar variability for zeta-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 zeta-potential of -22.8 +/- 0.8 mV and a tortuosity of 2.24 +/- 0.10.
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| 2. |
- Guy, Yifat, et al.
(författare)
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Determination of zeta-potential in rat organotypic hippocampal cultures.
- 2008
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Ingår i: Biophysical journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 94:11, s. 4561-9
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Tidskriftsartikel (refereegranskat)abstract
- zeta-potentials of entities such as cells and synaptosomes have been determined, but zeta of brain tissue has never been measured. Electroosmotic flow, and the resulting transport of neuroactive substances, would result from naturally occurring and experimentally or clinically induced electric fields if zeta is significant. We have developed a simple method for determining zeta in tissue. An electric field applied across a rat organotypic hippocampal slice culture (OHSC) drives fluorescent molecules through the tissue by both electroosmotic flow and electrophoresis. Fluorescence microscopy is used to determine each molecule's velocity. Independently, capillary electrophoresis is used to measure the molecules' electrophoretic mobilities. The experiment yields zeta-potential and average tissue tortuosity. The zeta-potential of OHSCs is -22 +/- 2 mV, and the average tortuosity is 1.83 +/- 0.06. In a refined experiment, zeta-potential is measured in various subregions. The zeta-potentials of the CA1 stratum pyramidale, CA3 stratum pyramidal, and dentate gyrus are -25.1 +/- 1.6 mV, -20.3 +/- 1.7 mV, and -25.4 +/- 1.0 mV, respectively. Simple dimensional arguments show that electroosmotic flow is potentially as important as diffusion in molecular transport.
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| 3. |
- Hamsher, Amy E, et al.
(författare)
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Minimizing tissue damage in electroosmotic sampling.
- 2010
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Ingår i: Analytical chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 82:15, s. 6370-6
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Tidskriftsartikel (refereegranskat)abstract
- Electroosmotic sampling is a potentially powerful method for pulling extracellular fluid into a fused-silica capillary in contact with the surface of tissue. An electric field is created in tissue by passing current through an electrolyte-filled capillary and then through the tissue. The resulting field acts on the counterions to the surface charges in the extracellular space to create electroosmotic fluid flow within the extracellular space of a tissue. Part of the development of this approach is to define conditions under which electroosmotic sampling minimizes damage to the tissue, in this case organotypic hippocampal slice cultures (OHSCs). We have assessed tissue damage by measuring fluorescence resulting from exposing sampled tissue to propidium iodide solution 16-24 h after sampling. Sampling has been carried out with a variety of capillary diameters, capillary tip-tissue distances, and applied voltages. Tissue damage is negligible when the power (current x potential drop) created in the tissue is less than 120 microW. In practical terms, smaller capillary i.d.s, lower voltages, and greater tissue to capillary distances lead to lower power.
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| 4. |
- Rupert, Amy E, et al.
(författare)
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A simple method for measuring organotypic tissue slice culture thickness.
- 2011
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Ingår i: Journal of neuroscience methods. - : Elsevier BV. - 1872-678X .- 0165-0270. ; 199:1, s. 78-81
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Tidskriftsartikel (refereegranskat)abstract
- This paper presents a simple method to measure tissue slice thicknesses using an ohmmeter. The circuit described here is composed of a metal probe, an ohmmeter, a counter electrode, culture medium or physiological buffer, and tissue slice. The probe and the electrode are on opposite interfaces of an organotypic hippocampal slice culture. The circuit closes when the metal probe makes contact with the surface of the tissue slice. The probe position is recorded and compared to its position when it makes contact with the insert membrane on which the tissue grows, thus yielding a thickness measurement. The method does not reduce the viability of slice cultures. Thicknesses of the slice cultures were measured under a number of culturing protocols. An initial drop in thickness occurred between 0 and 4 days in culture. Thicknesses are rather constant thereafter. The type of culture medium and the initial thickness of the tissue explant influence the thickness. Slice thicknesses were compared to a known technique by using optical measurements of slice cross-sections to obtain thicknesses. In contrast to this known technique, the proposed method does not sacrifice the slice culture for measurement purposes. The proposed measurement technique described is straightforward and rapid, about 1 min per culture.
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| 5. |
- Tranberg, Mattias, 1977, et al.
(författare)
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NMDA-receptor mediated efflux of N-acetylaspartate: physiological and/or pathological importance?
- 2004
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Ingår i: Neurochemistry international. - : Elsevier BV. - 0197-0186. ; 45:8, s. 1195-204
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Tidskriftsartikel (refereegranskat)abstract
- N-Acetylaspartate (NAA) is a largely neuron specific dianionic amino acid present in high concentration in vertebrate brain. Many fundamental questions concerning N-acetylaspartate in brain remain unanswered. One such issue is the predominantly neuronal synthesis and largely glial catabolism which implies the existence of a regulated efflux from neurons. Here we show that transient (5 min) NMDA-receptor activation (60 microM) induces a long lasting Ca2+ -dependent efflux of N-acetylaspartate from organotypic slices of rat hippocampus. The NMDA-receptor stimulated efflux was unaffected by hyper-osmotic conditions (120 mM sucrose) and no efflux of N-acetylaspartate was evoked by high K+ -depolarization (50 mM) or kainate (300 microM). These results indicate that the efflux induced by NMDA is not related directly to either cell swelling or depolarization but is coupled to Ca2+ -influx via the NMDA-receptor. The efflux of N-acetylaspartate persisted at least 20 min after the omission of NMDA, similar to the efflux of the organic anions glutathione and phosphoethanolamine. The efflux of taurine and hypotaurine was also stimulated by NMDA but returned more quickly to basal levels. The NMDA-receptor stimulated efflux of N-acetylaspartate, glutathione, phosphoethanolamine, taurine and hypotaurine correlated with delayed nerve cell death measured 24 h after the transient NMDA-receptor stimulation. However, exogenous administration of high concentrations of N-acetylaspartate to the culture medium was non-toxic. The results suggest that Ca2+ -influx via the NMDA-receptor regulates the efflux of N-acetylaspartate from neurons which may have both physiological and pathological importance.
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| 6. |
- Xu, Hongjuan, et al.
(författare)
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Electroosmotic sampling. Application to determination of ectopeptidase activity in organotypic hippocampal slice cultures.
- 2010
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Ingår i: Analytical chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 82:15, s. 6377-83
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Tidskriftsartikel (refereegranskat)abstract
- We hypothesize that peptide-containing solutions pulled through tissue should reveal the presence and activity of peptidases in the tissue. Using the natural zeta-potential in the organotypic hippocampal slice culture (OHSC), physiological fluids can be pulled through the tissue with an electric field. The hydrolysis of the peptides present in the fluid drawn through the tissue can be determined using capillary HPLC with electrochemical detection of the biuret complexes of the peptides following a postcolumn reaction. We have characterized this new sampling method by measuring the flow rate, examining the use of internal standards, and examining cell death caused by sampling. The sampling flow rate ranges from 60 to 150 nL/min with a 150 microm (ID) sampling capillary with an electric field (at the tip of the capillary) from 30 to 60 V/cm. Cell death can be negligible with controlled sampling conditions. Using this sampling approach, we have electroosmotically pulled Leu-enkephalin through OHSCs to identify ectopeptidase activity in the CA3 region. These studies show that a bestatin-sensitive aminopeptidase may be critical for the hydrolysis of exogenous Leu-enkephalin, a neuropeptide present in the CA3 region of OHSCs.
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