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Determination of zeta-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 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.

SUBMITTER: Guy Y

PROVIDER: S-EPMC2736137 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Determination of zeta-potential and tortuosity in rat organotypic hippocampal cultures from electroosmotic velocity measurements under feedback control.

Guy Yifat Y Muha Robert J RJ Sandberg Mats M Weber Stephen G SG

Analytical chemistry 20090401 8

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 diffe ...[more]

PMID: 19298057

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Project description:Elevated levels of glucocorticoids exert neurotoxic effects, and the hippocampus is particularly sensitive to the effects of glucocorticoids. Because some data have indicated that an increased action of glucocorticoids in the perinatal period enhances the susceptibility of brain tissue to adverse substances later in life, the main purpose of the present study was to compare necrotic/apoptotic corticosterone action in hippocampal organotypic cultures obtained from control animals with the effect of this steroid in tissue from prenatally stressed rats. Because the adverse effects of glucocorticoid action on nerve cell viability appear to result mainly from an increase in the intensity of the effects of glutamate and changes in growth factor and pro-inflammatory cytokine synthesis, the involvement of these factors in corticosterone action were also determined. In stress-like concentration (1 μM), corticosterone, when added to hippocampal cultures for 1 and 3 days, alone or jointly with glutamate, did not induce necrosis. In contrast, in 3-day cultures, corticosterone (1 μM) increased caspase-3 activity and the mRNA expression of the pro-apoptotic Bax. Moreover, corticosterone's effect on caspase-3 activity was stronger in hippocampal cultures from prenatally stressed compared to control rats. Additionally, 24 h of exposure to corticosterone and glutamate, when applied separately and together, increased Bdnf, Ngf, and Tnf-α expression. In contrast, after 72 h, a strong decrease in the expression of both growth factors was observed, while the expression of TNF-α remained high. The present study showed that in stress-like concentrations, corticosterone exerted pro-apoptotic but not necrotic effects in hippocampal organotypic cultures. Prenatal stress increased the pro-apoptotic effects of corticosterone. Increased synthesis of the pro-inflammatory cytokine TNF-α may be connected with the adverse effects of corticosterone on brain cell viability.

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Project description:Blast traumatic brain injury (bTBI) affects civilians, soldiers, and veterans worldwide and presents significant health concerns. The mechanisms of neurodegeneration following bTBI remain elusive and current therapies are largely ineffective. It is important to better characterize blast-evoked cellular changes and underlying mechanisms in order to develop more effective therapies. In the present study, our group utilized rat organotypic hippocampal slice cultures (OHCs) as an in vitro system to model bTBI. OHCs were exposed to either 138 ± 22 kPa (low) or 273 ± 23 kPa (high) overpressures using an open-ended helium-driven shock tube, or were assigned to sham control group. At 2 hours (h) following injury, we have characterized the astrocytic response to a blast overpressure. Immunostaining against the astrocytic marker glial fibrillary acidic protein (GFAP) revealed acute shearing and morphological changes in astrocytes, including clasmatodendrosis. Moreover, overlap of GFAP immunostaining and propidium iodide (PI) indicated astrocytic death. Quantification of the number of dead astrocytes per counting area in the hippocampal cornu Ammonis 1 region (CA1), demonstrated a significant increase in dead astrocytes in the low- and high-blast, compared to sham control OHCs. However only a small number of GFAP-expressing astrocytes were co-labeled with the apoptotic marker Annexin V, suggesting necrosis as the primary type of cell death in the acute phase following blast exposure. Moreover, western blot analyses revealed calpain mediated breakdown of GFAP. The dextran exclusion additionally indicated membrane disruption as a potential mechanism of acute astrocytic death. Furthermore, although blast exposure did not evoke significant changes in glutamate transporter 1 (GLT-1) expression, loss of GLT-1-expressing astrocytes suggests dysregulation of glutamate uptake following injury. Our data illustrate the profound effect of blast overpressure on astrocytes in OHCs at 2 h following injury and suggest increased calpain activity and membrane disruption as potential underlying mechanisms.

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Determination of zeta-potential and tortuosity in rat organotypic hippocampal cultures from electroosmotic velocity measurements under feedback control.

Publications

Determination of zeta-potential and tortuosity in rat organotypic hippocampal cultures from electroosmotic velocity measurements under feedback control.

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