Biomedical subjects

Alexandr Chvátal

Publications and source records attributed to Alexandr Chvátal.

5 recordsLinked to original sources

PubMed2007

Three-dimensional confocal morphometry reveals structural changes in astrocyte morphology in situ.

Neuronal activity and many pathological states in the CNS are accompanied by transient astrocytic swelling, which affects excitability, extrasynaptic transmission, and neuron-glia interactions. By using three-dimensional confocal morphometry (3DCM), we quantified the morphometric parameters of astrocytes in intact tissue. In experiments performed in brain cortex slices from transgenic GFAP/EGFP mice, we applied 3DCM to study the dynamic changes in astrocyte morphology during hypotonic stress. Our morphometric analysis showed that the effect of a 10-min application of hypotonic solution (200 mmol/kg) on the swelling of different cell compartments was dependent on the extent of the swelling of the total astrocyte volume. If the swelling of the whole cell, i.e., soma and processes, was less than approximately 10%, there were no differences between the swelling of the soma and the processes. However, if the swelling of the total cell volume was greater than 10%, the swelling of the processes was greater than the swelling of the soma. Analyzing the effect of hypotonic solution on the morphology of these astrocytes revealed that the total cell volume increased; however, certain cell compartments were distinguished in which the volume increased, whereas in other compartments cell volume decreased or apparently did not change, and the structure of some compartments was altered. Our data show that astrocytes in brain slices undergoing hypotonic stress display cell volume regulation as well as transient changes in morphology.

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PubMed2006

Transplantation of embryonic neuroectodermal progenitor cells into the site of a photochemical lesion: immunohistochemical and electrophysiological analysis.

GFP labeled/NE-4C neural progenitor cells cloned from primary neuroectodermal cultures of p53- mouse embryos give rise to neurons when exposed to retinoic acid in vitro. To study their survival and differentiation in vivo, cells were transplanted into the cortex of 6-week-old rats, 1 week after the induction of a photochemical lesion or into noninjured cortex. The electrophysiological properties of GFP/NE-4C cells were studied in vitro (8-10 days after differentiation induction) and 4 weeks after transplantation using the whole-cell patch-clamp technique, and immunohistochemical analyses were carried out. After transplantation into a photochemical lesion, a large number of cells survived, some of which expressed the astrocytic marker GFAP. GFP/GFAP-positive cells, with an average resting membrane potential (Vrest) of -71.9 mV, displayed passive time- and voltage-independent K+ currents and, additionally, voltage-dependent A-type K+ currents (KA) and/or delayed outwardly rectifying K+ currents (KDR). Numerous GFP-positive cells expressed NeuN, betaIII-tubulin, or 68 kD neurofilaments. GFP/betaIII-tubulin-positive cells, with an average Vrest of -61.6 mV, were characterized by the expression of KA and KDR currents and tetrodotoxin-sensitive Na+ currents. GFP/NE-4C cells also gave rise to oligodendrocytes, based on the detection of oligodendrocyte-specific markers. Our results indicate that GFP/NE-4C neural progenitors transplanted into the site of a photochemical lesion give rise to neurons and astrocytes with membrane properties comparable to those transplanted into noninjured cortex. Therefore, GFP/NE-4C cells provide a suitable model for studying neuro- and gliogenesis in vivo. Further, our results suggest that embryonic neuroectodermal progenitor cells may hold considerable promise for the repair of ischemic brain lesions.

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PubMed2004

Analysis of K+ accumulation reveals privileged extracellular region in the vicinity of glial cells in situ.

Astrocytes and oligodendrocytes in rat and mouse spinal cord slices, characterized by passive membrane currents during de- and hyperpolarizing stimulation pulses, express a high resting K+ conductance. In contrast to the case for astrocytes, a depolarizing prepulse in oligodendrocytes produces a significant shift of reversal potential (Vrev) to positive values, arising from the larger accumulation of K+ in the vicinity of the oligodendrocyte membrane. As a result, oligodendrocytes express large tail currents (Itail) after a depolarizing prepulse due to the shift of K+ into the cell. In the present study, we used a mathematical model to calculate the volume of the extracellular space (ECS) in the vicinity of astrocytes and oligodendrocytes (ESVv), defined as the volume available for K+ accumulation during membrane depolarization. A mathematical analysis of membrane currents revealed no differences between glial cells from mouse (n = 59) or rat (n = 60) spinal cord slices. We found that the Vrev of a cell after a depolarizing pulse increases with increasing Itail, expressed as the ratio of the integral inward current (Qin) after the depolarizing pulse to the total integral outward current (Qout) during the pulse. In astrocytes with small Itail and Vrev ranging from -50 to -70 mV, the Qin was only 3-19% of Qout, whereas, in oligodendrocytes with large Itail and Vrev between -20 and 0 mV, Qin/Qout was 30-75%. On the other hand, ESVv decreased with increasing values of Vrev. In astrocytes, ESVv ranged from 2 to 50 microm3, and, in oligodendrocytes, it ranged from 0.1 to 2.0 microm3. Cell swelling evoked by the application of hypotonic solution shifted Vrev to more positive values by 17.2 +/- 1.8 mV and was accompanied by a decrease in ESVv of 3.6 +/- 1.3 microm3. Our mathematical analysis reveals a 10-100 times smaller region of the extracellular space available for K+ accumulation during cell depolarization in the vicinity of oligodendrocytes than in the vicinity of astrocytes. The presence of such privileged regions around cells in the CNS may affect the accumulation and diffusion of other neuroactive substances and alter communication between cells in the CNS.

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PubMed2004

Voltage-dependent potassium currents in hypertrophied rat astrocytes after a cortical stab wound.

Changes in the membrane properties of reactive astrocytes in gliotic cortex induced by a stab wound were studied in brain slices of 21-28-day-old rats, using the patch-clamp technique and were correlated with changes in resting extracellular K+ concentration ([K+]e) measured in vivo using K+-selective microelectrodes. Based on K+ current expression, three types of astrocytes were identified in gliotic cortex: A1 astrocytes expressing a time- and voltage-independent K+ current component and additional inwardly rectifying K+ currents (K(IR)); A2 astrocytes expressing a time- and voltage-independent K+ current component and additional delayed outwardly rectifying K+ currents (K(DR)); and complex astrocytes expressing K(DR), K(IR), and A-type K+ (K(A)) currents and Na+ currents (I(Na)). Nestin/bromodeoxyuridine (BrdU)-negative A1 astrocytes were found further than approximately 100 microm from the stab wound and showed an upregulation of K(IR) currents within the first day post-injury (PI), correlating with an increased resting [K+]e. Their number declined from 62% of total astrocytes in control rats to 41% in rats at 7 days PI. Nestin/BrdU-positive A2 astrocytes were found only within a distance of approximately 100 microm from the stab wound and, in comparison to those in control rats, showed an upregulation of K(DR) currents. Their number increased from 8% of the total number of astrocytes in control rats to 39% 7 days PI. Both A1 and A2 astrocytes showed hypertrophied processes and increased GFAP staining, but an examination of cell morphology revealed greater changes in the surface/volume ratio in A2 astrocytes than in A1 astrocytes. Complex astrocytes did not display a hypertophied morphology; K(IR) currents in these cells were upregulated within 1 day PI, while the K(DR), K(A), and I(Na) currents were increased only 6 h PI. We conclude that two electrophysiologically, immunohistochemically, and morphologically distinct types of hypertrophied astrocytes are present at the site of a stab wound, depending on the distance from the lesion, and may have different functions in ionic homeostasis and/or regeneration.

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PubMed2003

The relationship between changes in intrinsic optical signals and cell swelling in rat spinal cord slices.

Changes in intrinsic optical signals could be related to cell swelling; however, the evidence is not compelling. We measured light transmittance, ECS volume fraction (alpha), and extracellular K+ in rat spinal cord slices during electrical stimulation and the application of elevated potassium, NMDA, or anisoosmotic solutions. Dorsal root stimulation (10 Hz/1 min) induced an elevation in extracellular K+ to 6-8 mM, a light transmittance increase of 6-8%, and a relative ECS volume decrease of less than 5%; all of these changes had different time courses. The application of 6 or 10 mM K+ or NMDA (10(-5) M) had no measurable effect on alpha, but light transmittance increased by 20-25%. The application of 50 or 80 mM K+ evoked a 72% decrease in alpha while the light transmittance increase remained as large as that in 6 or 10 mM K+. While the change in alpha persisted throughout the 45-min application, light transmittance, after peaking in 6-8 min, quickly returned to control levels and decreased below them. Astrocytic hypertrophy was observed in 6, 10, and 50 mM K+. The same results followed the application of 10(-4) M NMDA or hypotonic solution (160 mmol/kg). The elevation of extracellular K+ after NMDA application, corresponding to increased neuronal activity, had a similar time course as the light transmittance changes. Furosemide, Cl(-)-free, or Ca(2+)-free solution blocked or slowed down the decreases in alpha, while the light transmittance increases were unaffected. In hypertonic solution (400 mmol/kg), alpha increased by 30-40%, while light transmittance decreased by 15-20%. Thus, light transmittance changes do not correlate with changes in ECS volume but are associated with neuronal activity and morphological changes in astrocytes.

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