Biomedical subjects

Eva Syková

Publications and source records attributed to Eva Syková.

At least 19 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.

Animals↗

PubMed2006

Brain metabolism and extracellular space diffusion parameters during and after transient global hypoxia in the rat cortex.

Hypoxia results in both reversible and irreversible changes in the brain extracellular space (ECS). This study utilized microdialysis to monitor changes in the energy-related metabolites lactate, pyruvate, glucose and glutamate in the rat cortex before, during and after 30-min transient global hypoxia, induced in anesthetized rats by reducing inspired oxygen to 6% O(2) in nitrogen. Changes in metabolite levels were compared with ECS diffusion parameters calculated from diffusion curves of tetramethylammonium applied by iontophoresis. Significant increases in lactate concentration and the lactate/pyruvate ratio, as well as decreased glucose levels, were found in the cortex immediately after the induction of hypoxia. Following recovery to ventilation with air, extracellular lactate and glucose levels and the lactate/pyruvate ratio returned to control levels within 40, 20 and 30 min, respectively. Glutamate levels started to increase 20-30 min after the onset of hypoxia and returned to prehypoxic values within 30-40 min of reoxygenation. The ECS volume fraction alpha decreased by about 5% from 0.18+/-0.01 during the first 20-25 min of hypoxia; after 25 min alpha dropped a further 22% to 0.14+/-0.01. Within 10 min of reoxygenation, alpha returned to control values, then increased to 0.20+/-0.01 and remained at this level until the end of the experiment. The observed 22% decrease in alpha markedly influences dialysate levels measured during hypoxia. In our study, the complete posthypoxic recovery of cortical metabolite levels and ECS diffusion properties suggests that metabolic enzymes and related cellular components (e.g., mitochondria) may tolerate prolonged hypoxic periods and recover to prehypoxic values.

Animals↗

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.

Animals↗

PubMed2006

Changes in diffusion parameters, energy-related metabolites and glutamate in the rat cortex after transient hypoxia/ischemia.

It has been shown that global anoxia leads to dramatic changes in the diffusion properties of the extracellular space (ECS). In this study, we investigated how changes in ECS volume and geometry in the rat somatosensory cortex during and after transient hypoxia/ischemia correlate with extracellular concentrations of energy-related metabolites and glutamate. Adult male Wistar rats (n = 12) were anesthetized and subjected to hypoxia/ischemia for 30 min (ventilation with 10% oxygen and unilateral carotid artery occlusion). The ECS diffusion parameters, volume fraction and tortuosity, were determined from concentration-time profiles of tetramethylammonium applied by iontophoresis. Concentrations of lactate, glucose, pyruvate and glutamate in the extracellular fluid (ECF) were monitored by microdialysis (n = 9). During hypoxia/ischemia, the ECS volume fraction decreased from initial values of 0.19 +/- 0.03 (mean +/- S.E.M.) to 0.07 +/- 0.01 and tortuosity increased from 1.57 +/- 0.01 to 1.88 +/- 0.03. During reperfusion the volume fraction returned to control values within 20 min and then increased to 0.23 +/- 0.01, while tortuosity only returned to original values (1.53 +/- 0.06). The concentrations of lactate and glutamate, and the lactate/pyruvate ratio, substantially increased during hypoxia/ischemia, followed by continuous recovery during reperfusion. The glucose concentration decreased rapidly during hypoxia/ischemia with a subsequent return to control values within 20 min of reperfusion. We conclude that transient hypoxia/ischemia causes similar changes in ECS diffusion parameters as does global anoxia and that the time course of the reduction in ECS volume fraction correlates with the increase of extracellular concentration of glutamate. The decrease in the ECS volume fraction can therefore contribute to an increased accumulation of toxic metabolites, which may aggravate functional deficits and lead to damage of the central nervous system (CNS).

Animals↗

PubMed2006

Bone marrow stem cells and polymer hydrogels--two strategies for spinal cord injury repair.

1. Emerging clinical studies of treating brain and spinal cord injury (SCI) led us to examine the effect of autologous adult stem cell transplantation as well as the use of polymer scaffolds in spinal cord regeneration. We compared an intravenous injection of mesenchymal stem cells (MSCs) or the injection of a freshly prepared mononuclear fraction of bone marrow cells (BMCs) on the treatment of an acute or chronic balloon-induced spinal cord compression lesion in rats. Based on our experimental studies, autologous BMC implantation has been used in a Phase I/II clinical trial in patients (n=20) with a transversal spinal cord lesion. 2. MSCs were isolated from rat bone marrow by their adherence to plastic, labeled with iron-oxide nanoparticles and expanded in vitro. Macroporous hydrogels based on derivatives of 2-hydroxyethyl methacrylate (HEMA) or 2-hydroxypropyl methacrylamide (HPMA) were prepared, then modified by their copolymerization with a hydrolytically degradable crosslinker, N,O-dimethacryloylhydroxylamine, or by different surface electric charges. Hydrogels or hydrogels seeded with MSCs were implanted into rats with hemisected spinal cords. 3. Lesioned animals grafted with MSCs or BMCs had smaller lesions 35 days postgrafting and higher scores in BBB testing than did control animals and also showed a faster recovery of sensitivity in their hind limbs using the plantar test. The functional improvement was more pronounced in MSC-treated rats. In MR images, the lesion populated by grafted cells appeared as a dark hypointense area and was considerably smaller than in control animals. Morphometric measurements showed an increase in the volume of spared white matter in cell-treated animals. In the clinical trial, we compared intraarterial (via a. vertebralis, n=6) versus intravenous administration of BMCs (n=14) in a group of subacute (10-33 days post-SCI, n=8) and chronic patients (2-18 months, n=12). For patient follow-up we used MEP, SEP, MRI, and the ASIA score. Our clinical study revealed that the implantation of BMCs into patients is safe, as there were no complications following cell administration. Partial improvement in the ASIA score and partial recovery of MEP or SEP have been observed in all subacute patients who received cells via a. vertebralis (n=4) and in one out of four subacute patients who received cells intravenously. Improvement was also found in one chronic patient who received cells via a. vertebralis. A much larger population of patients is needed before any conclusions can be drawn. The implantation of hydrogels into hemisected rat spinal cords showed that cellular ingrowth was most pronounced in copolymers of HEMA with a positive surface electric charge. Although most of the cells had the morphological properties of connective tissue elements, we found NF-160-positive axons invading all the implanted hydrogels from both the proximal and distal stumps. The biodegradable hydrogels degraded from the border that was in direct contact with the spinal cord tissue. They were resorbed by macrophages and replaced by newly formed tissue containing connective tissue elements, blood vessels, GFAP-positive astrocytic processes, and NF-160-positive neurofilaments. Additionally, we implanted hydrogels seeded with nanoparticle-labeled MSCs into hemisected rat spinal cords. Hydrogels seeded with MSCs were visible on MR images as hypointense areas, and subsequent Prussian blue histological staining confirmed positively stained cells within the hydrogels. 4. We conclude that treatment with different bone marrow cell populations had a positive effect on behavioral outcome and histopathological assessment after SCI in rats; this positive effect was most pronounced following MSC treatment. Our clinical study suggests a possible positive effect in patients with SCI. Bridging the lesion cavity can be an approach for further improving regeneration. Our preclinical studies showed that macroporous polymer hydrogels based on derivatives of HEMA or HPMA are suitable materials for bridging cavities after SCI; their chemical and physical properties can be modified to a specific use, and 3D implants seeded with different cell types may facilitate the ingrowth of axons.

Animals↗

PubMed2006

Hypoosmolar conditions reduce extracellular volume fraction and enhance epileptiform activity in the CA3 region of the immature rat hippocampus.

The osmolarity of the extracellular space (ECS) compartment is an important factor determining the excitability of neuronal tissue. In the adult hippocampus an important role of osmolarity and ECS diffusion parameters on the susceptibility to epileptic events is well established, but the influence of hypo- and hyperosmolar conditions on the immature hippocampus remains elusive. To investigate the influence of osmolarity on epileptiform activity, extracellular field potentials were recorded in the CA3 region of hippocampal slices of immature (postnatal days 4-7) Wistar rats. The ECS diffusion parameters were determined by the real-time tetramethylammonium (TMA+) iontophoretic method with ion-selective microelectrodes in immature hippocampal slices and showed a lack of diffusion anisotropy; a tortuosity of about 1.39; and a volume fraction, alpha, of 0.41 +/- 0.01 (n = 10 slices). A reduction in osmolarity of -90 mOsm induced a decrease in alpha to 0.17 +/- 0.02 (n = 4 slices). The frequency of epileptiform activity elicited in 10-50 microM 4-AP-containing low-Mg2+ solution was increased under -90 mOsm and -40 mOsm hypoosmolar conditions by 39.9% +/- 8.1% (n = 16) and 24.1% +/- 4.8% (n = 10), respectively, whereas hyperosmolar solutions decreased the frequency. A -90-mOsm reduction in the osmolarity of low-Mg2+ solution induced epileptiform activity in nine of 19 slices. In summary, these results demonstrate that hypoosmolar conditions increased excitability and susceptibility to epileptiform activity in immature hippocampal slices, suggesting a functional role of the larger alpha in suppression of seizures.

4-Aminopyridine↗

PubMed2006

Macroporous hydrogels based on 2-hydroxyethyl methacrylate. Part 5: hydrolytically degradable materials.

Macroporous hydrogels based on 2-hydroxyethyl methacrylate, 2-ethoxyethyl methacrylate and N-(2-hydroxypropyl)methacrylamide, methacrylic acid and [2-(methacryloyloxy)ethyl]trimethylammonium chloride crosslinked with N,O-dimethacryloylhydroxylamine were prepared. Hydrogels were degraded in a buffer of pH 7.4. Completely water-soluble polymers were obtained over time periods ranging from 2 to 40 days. The process of degradation was followed gravimetrically and by optical and electron microscopy. In vivo biological tests with hydrogels based on copolymers of 2-ethoxyethyl methacrylate/N-(2-hydroxypropyl)methacrylamide were performed.

Absorbable Implants↗

PubMed2006

Transplantation of bone marrow stem cells as well as mobilization by granulocyte-colony stimulating factor promotes recovery after spinal cord injury in rats.

Emerging clinical studies of treating brain and spinal cord injury (SCI) with autologous adult stem cells led us to compare the effect of an intravenous injection of mesenchymal stem cells (MSCs), an injection of a freshly prepared mononuclear fraction of bone marrow cells (BMCs) or bone marrow cell mobilization induced by granulocyte colony stimulating factor (G-CSF) in rats with a balloon- induced spinal cord compression lesion. MSCs were isolated from rat bone marrow by their adherence to plastic, labeled with iron-oxide nanoparticles and expanded in vitro. Seven days after injury, rats received an intravenous injection of MSCs or BMCs or a subcutaneous injection of GCSF (from day 7 to 11 post-injury). Functional status was assessed weekly for 5 weeks after SCI, using the Basso-Beattie-Bresnehan (BBB) locomotor rating score and the plantar test. Animals with SCI treated with MSCs, BMCs, or G-CSF had higher BBB scores and better recovery of hind limb sensitivity than controls injected with saline. Morphometric measurements showed an increase in the spared white matter. MR images of the spinal cords were taken ex vivo 5 weeks after SCI using a Bruker 4.7-T spectrometer. The lesions populated by grafted MSCs appeared as dark hypointense areas. Histology confirmed a large number of iron-containing and PKH 26-positive cells in the lesion site. We conclude that treatment with three different bone marrow cell populations had a positive effect on behavioral outcome and histopathological assessment after SCI, which was most pronounced after MSC injection.

Animals↗

PubMed2006

Magnetic resonance tracking of transplanted stem cells in rat brain and spinal cord.

BACKGROUND: Magnetic resonance imaging provides a noninvasive method to study the fate of transplanted cells in vivo. OBJECTIVE: We studied the fate of embryonic and mesenchymal stem cells labeled with iron oxide nanoparticles (Endorem) and human CD34+ cells labeled with magnetic MicroBeads (Miltenyi) in rats with a cortical or spinal cord lesion. METHODS: Cells were grafted intracerebrally, contralaterally to a cortical photochemical lesion, or injected intravenously. RESULTS: During the 1st week after transplantation, transplanted cells migrated to the lesion and were visible in the lesion on MR images as a hypointensive signal, persisting for more than 30 days. In rats with a balloon-induced spinal cord compression lesion, we observed an increase in functional recovery and hind limb sensitivity after implantation of Endorem-labeled mesenchymal stem cells or a freshly prepared mononuclear fraction of bone marrow cells or after injection of granulocyte colony-stimulating factor. Morphometric measurements in the center of the lesions showed an increase in white matter volume in cell-treated animals. Prussian blue staining confirmed a large number of Prussian blue-positive cells, and the lesions were considerably smaller than in control animals. CONCLUSIONS: These studies demonstrate that magnetic resonance imaging of grafted adult as well as embryonic stem cells labeled with iron oxide nanoparticles is a useful method for evaluating their migration and fate in CNS.

Animals↗

PubMed2005

Decreased proliferation in the adult rat hippocampus after exposure to the Morris water maze and its reversal by fluoxetine.

Granular cell proliferation in the adult hippocampus decreases during aging and after chronic stress, while it can be increased by physical activity or treatment with the antidepressant fluoxetine. We investigated whether the physical and cognitive stimulation accompanied by stress in the commonly used Morris water maze affects the rate of proliferation and whether the induced changes can be influenced by antidepressant treatment with fluoxetine. Proliferating cells in the dentate gyrus were labeled by three injections of BrdU during the 24h preceding sacrifice. Early differentiation to neuronal progeny was studied by immunohistochemical staining for doublecortin (DCX), a microtubule binding protein expressed in newborn neurons. Acquisition learning in the water maze for 15 days caused a significant decrease in granular cell proliferation in the granular cell layer of the hippocampus. The decrease in the number of BrdU- and DCX-positive cells was reversed to control levels by the use of fluoxetine during the water maze training. Fluoxetine treatment alone increased the number of BrdU-positive cells, but did not increase the number of DCX-positive cells. We conclude that the exposure of adult male rats to water maze acquisition trials is a stressful experience that significantly suppresses the production of new granular cells and that this stressful effect can be blocked by the concomitant administration of the antidepressant fluoxetine.

Adaptation, Physiological↗

PubMed2005

Changes in extracellular space size and geometry in APP23 transgenic mice: a model of Alzheimer's disease.

Diffusion parameters of the extracellular space (ECS) are changed in many brain pathologies, disturbing synaptic as well as extrasynaptic "volume" transmission, which is based on the diffusion of neuroactive substances in the ECS. Amyloid deposition, neuronal loss, and disturbed synaptic transmission are considered to be the main causes of Alzheimer's disease dementia. We studied diffusion parameters in the cerebral cortex of transgenic APP23 mice, which develop a pathology similar to Alzheimer's disease. The real-time tetramethylammonium (TMA) method and diffusion-weighted MRI were used to measure the ECS volume fraction (alpha = ECS volume/total tissue volume) and the apparent diffusion coefficients (ADCs) of TMA (ADC(TMA)), diffusing exclusively in the ECS and of water (ADC(W)). Measurements were performed in vivo in 6-, 8-, and 17- to 25-month-old hemizygous APP23 male and female mice and age-matched controls. In all 6- to 8-month-old APP23 mice, the mean ECS volume fraction, ADC(TMA), and ADC(W) were not significantly different from age-matched controls (alpha = 0.20 +/- 0.01; ADC(TMA), 580 +/- 16 microm(2).s(-1); ADC(W), 618 +/- 19 microm(2).s(-1)). Aging in 17- to 25-month-old controls was accompanied by a decrease in ECS volume fraction and ADC(W), significantly greater in females than in males, but no changes in ADC(TMA). ECS volume fraction increased (0.22 +/- 0.01) and ADC(TMA) decreased (560 +/- 7 microm(2).s(-1)) in aged APP23 mice. The impaired navigation observed in these animals in the Morris water maze correlated with their plaque load, which was twice as high in females (20%) as in males (10%) and may, together with changed ECS diffusion properties, account for the impaired extrasynaptic transmission and spatial cognition observed in old transgenic females.

Aging↗

PubMed2005

Reduced extracellular space in the brain of tenascin-R- and HNK-1-sulphotransferase deficient mice.

Tenascin-R (TN-R), a large extracellular glycoprotein, is an important component of the adult brain's extracellular matrix (ECM); tenascin-C (TN-C) is expressed mainly during early development, while human natural killer 1 (HNK-1) is a sulphated carbohydrate epitope that attaches to these molecules, modifying their adhesive properties. To assess their influence on extracellular space (ECS) volume and geometry, we used the real-time iontophoretic method to measure ECS volume fraction alpha and tortuosity lambda, and diffusion-weighted magnetic resonance imaging (MRI) to measure the apparent diffusion coefficient of water (ADC(W)). Measurements were performed in vivo in the cortex and CA1 hippocampal region of TN-R-, TN-C- and HNK-1 sulphotransferase (ST)-deficient adult mice and their wild-type littermate controls. In both cortex and hippocampus, the lack of TN-R or HNK-1 sulphotransferase resulted in a significant decrease in alpha and lambda. Compared with controls, alpha in TN-R-/- and ST-/- mice decreased by 22-26% and 9-15%, respectively. MRI measurements revealed a decreased ADC(W) in the cortex, hippocampus and thalamus. ADC(W) reflected the changes in alpha; the decrease in lambda indicated fewer diffusion obstacles in the ECS, presumably due to a decreased macromolecular content. No significant changes were found in TN-C-/- animals. We conclude that in TN-R-/- and ST-/- mice, which show morphological, electrophysiological and behavioural abnormalities, the ECS is reduced and its geometry altered. TN-R, as an important component of the ECM, appears to maintain an optimal distance between cells. The altered diffusion of neuroactive substances in the brain will inevitably affect extrasynaptic transmission, neuron-glia interactions and synaptic efficacy.

Animals↗

PubMed2005

Magnetic resonance tracking of implanted adult and embryonic stem cells in injured brain and spinal cord.

Stem cells are a promising tool for treating brain and spinal cord injury. Magnetic resonance imaging (MRI) provides a noninvasive method to study the fate of transplanted cells in vivo. We studied implanted rat bone marrow stromal cells (MSCs) and mouse embryonic stem cells (ESCs) labeled with iron-oxide nanoparticles (Endorem) and human CD34+ cells labeled with magnetic MicroBeads (Miltenyi) in rats with a cortical or spinal cord lesion. Cells were grafted intracerebrally, contralaterally to a cortical photochemical lesion, or injected intravenously. During the first week post transplantation, transplanted cells migrated to the lesion. About 3% of MSCs and ESCs differentiated into neurons, while no MSCs, but 75% of ESCs differentiated into astrocytes. Labeled MSCs, ESCs, and CD34+ cells were visible in the lesion on MR images as a hypointensive signal, persisting for more than 50 days. In rats with a balloon-induced spinal cord compression lesion, intravenously injected MSCs migrated to the lesion, leading to a hypointensive MRI signal. In plantar and Basso-Beattie-Bresnehan (BBB) tests, grafted animals scored better than lesioned animals injected with saline solution. Histologic studies confirmed a decrease in lesion size. We also used 3-D polymer constructs seeded with MSCs to bridge a spinal cord lesion. Our studies demonstrate that grafted adult as well as embryonic stem cells labeled with iron-oxide nanoparticles migrate into a lesion site in brain as well as in spinal cord.

Animals↗

PubMed2005

Magnetic resonance tracking of human CD34+ progenitor cells separated by means of immunomagnetic selection and transplanted into injured rat brain.

Magnetic resonance imaging (MRI) provides a noninvasive method for studying the fate of transplanted cells in vivo. We studied whether superparamagnetic nanoparticles (CD34 microbeads), used clinically for specific magnetic sorting, can be used as a magnetic cell label for in vivo cell visualization. Human cells from peripheral blood were selected by CliniMACS CD34 Selection Technology (Miltenyi). Purified CD34+ cells were implanted into rats with a cortical photochemical lesion, contralaterally to the lesion. Twenty-four hours after grafting, the implanted cells were detected in the contralateral hemisphere as a hypointense spot on T2 weighted images; the hypointensity of the implant decreased during the first week. At the lesion site we observed a hypointensive signal 10 days after grafting that persisted for the next 3 weeks, until the end of the experiment. Prussian blue and anti-human nuclei staining confirmed the presence of magnetically labeled human cells in the corpus callosum and in the lesion 4 weeks after grafting. CD34+ cells were also found in the subventricular zone (SVZ). Human DNA (a human-specific 850 base pair fragment of alpha-satellite DNA from human chromosome 17) was detected in brain tissue sections from the lesion using PCR, confirming the presence of human cells. Our results show that CD34 microbeads superparamagnetic nanoparticles can be used as a magnetic cell label for in vivo cell visualization. The fact that microbeads coated with different commercially available antibodies can bind to specific cell types opens extensive possibilities for cell tracking in vivo.

Animals↗

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.

Animals↗

PubMed2004

Glutamate-evoked alterations of glial and neuronal cell morphology in the guinea pig retina.

Neuronal activity is accompanied by transmembranous ion fluxes that cause cell volume changes. In whole mounts of the guinea pig retina, application of glutamate resulted in fast swelling of neuronal cell bodies in the ganglion cell layer (GCL) and the inner nuclear layer (INL) (by approximately 40%) and a concomitant decrease of the thickness of glial cell processes in the inner plexiform layer (IPL) (by approximately 40%) that was accompanied by an elongation of the glial cells, by a thickening of the whole retinal tissue, and by a shrinkage of the extracellular space (by approximately 18%). The half-maximal effect of glutamate was observed at approximately 250 mum, after approximately 4 min. The swelling was caused predominantly by AMPA-kainate receptor-mediated influx of Na+ into retinal neurons. Similar but transient morphological alterations were induced by high K+ and dopamine, which caused release of endogenous glutamate and subsequent activation of AMPA-kainate receptors. Apparently, retinal glutamatergic transmission is accompanied by neuronal cell swelling that causes compensatory morphological alterations of glial cells. The effect of dopamine was elicitable only during light adaptation but not in the dark, and glutamate and high K+ induced strong ereffects in the dark than in the light. This suggests that not only the endogenous release of dopamine but also the responsiveness of glutamatergic neurons to dopamine is regulated by light-dark adaptation. Similar morphological alterations (neuronal swelling and decreased glial process thickness) were observed in whole mounts isolated immediately after experimental retinal ischemia, suggesting an involvement of AMPA-kainate receptor activation in putative neurotoxic cell swelling in the postischemic retina.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2004

Temporal profile of ultrastructural changes in cortical neurons after a photochemical lesion.

A photochemical lesion was induced in the right sensory motor cortex of rat brains. We examined at various time points the occurrence of different types of neuronal death with respect to a potential therapeutic window. The lesion appearance was documented by magnetic resonance imaging, and functional recovery was evaluated by behavioral tests showing recovery at 48 hr after lesioning. At 0.5, 1, 3, 6, 12, 24, 48, and 72 hr postlesion, cortical layers IV and V were examined by light and electron microscopy. Ultrastructural changes, which corresponded well to light microscopy findings, were found in both hemispheres. In the lesioned area, the neuropil appeared disorganized at 0.5 hr, and apoptotic and necrotic cell death was found at 0.5-3 hr. After 3 hr, the tissue was disintegrated. On the contralateral side, chromatin clumping appeared at 0.5-3 hr. At 3 hr, ruptured membranes were found, a sign of irreversible cell death. At 6-72 hr, the membranes were intact, and the chromatin was not clumped but heterogeneously distributed. The nuclei contained dispersed nucleoli at 48-72 hr. The morphology correlated well with magnetic resonance images and functional behavior. Our study demonstrates that a photochemical lesion is a useful model for studying morphological changes in injured cells. It results in a permanent infarction within 3 hr. In that the morphology on the contralateral side drastically changed between 3 and 6 hr, the cellular alterations at these time points might represent a break point at which cells either progress toward cell death or recover.

Animals↗

PubMed2004

Magnetic resonance tracking of transplanted bone marrow and embryonic stem cells labeled by iron oxide nanoparticles in rat brain and spinal cord.

Nuclear magnetic resonance (MR) imaging provides a noninvasive method for studying the fate of transplanted cells in vivo. We studied, in animals with a cortical photochemical lesion or with a balloon-induced spinal cord compression lesion, the fate of implanted rat bone marrow stromal cells (MSCs) and mouse embryonic stem cells (ESCs) labeled with superparamagnetic iron oxide nanoparticles (Endorem). MSCs were colabeled with bromodeoxyuridine (BrdU), and ESCs were transfected with pEGFP-C1 (eGFP ESCs). Cells were either grafted intracerebrally into the contralateral hemisphere of the adult rat brain or injected intravenously. In vivo MR imaging was used to track their fate; Prussian blue staining and electron microscopy confirmed the presence of iron oxide nanoparticles inside the cells. During the first week postimplantation, grafted cells migrated to the lesion site and populated the border zone of the lesion. Less than 3% of MSCs differentiated into neurons and none into astrocytes; 5% of eGFP ESCs differentiated into neurons, whereas 70% of eGFP ESCs became astrocytes. The implanted cells were visible on MR images as a hypointense area at the injection site, in the corpus callosum and in the lesion. The hypointense signal persisted for more than 50 days. The presence of GFP-positive or BrdU-positive and nanoparticle-labeled cells was confirmed by histological staining. Our study demonstrates that both grafted MSCs and eGFP ESCs labeled with a contrast agent based on iron oxide nanoparticles migrate into the injured CNS. Iron oxide nanoparticles can therefore be used as a marker for the long-term noninvasive MR tracking of implanted stem cells.

Animals↗