BACKGROUND: Several techniques for cell volume measurement using fluorescence microscopy have been established to date. In this study, we compare the performance of three different approaches which allow for estimations of the cell volume changes in biological samples containing individual fluorescently labeled cells either in culture or in the tissue context. The specific requirements, limitations and advantages of individual approaches are discussed. NEW METHOD: Global morphometric data are quantitatively compared with local information about the overall cell volume, represented by the concentration of a mobile fluorophore accumulated within the monitored cell. RESULTS: Volume changes induced by variations in the extracellular osmolarity in murine fibroblasts and astrocytes either in the culture or in the acute brain slices were registered by the three- and two-dimensional morphometries and by local fluorescence intensity measurements. The performance of the latter approach was verified using FRAP assessment of the fluorophore mobility. Significantly lower amplitudes of the cortical astrocytes swelling were detected by three-dimensional morphometry, when compared to the other two approaches. Consequently, it failed to detect temperature-induced cell volume changes. COMPARISON WITH EXISTING METHOD(S): The three most popular methods of cell volume measurement are compared to each other in this study. CONCLUSIONS: We show that the effectivity of global morphometry-based volumetric approaches drops with the increasing cell shape complexity or in the tissue context. In contrast to this, the performance of local fluorescence intensity monitoring, which is also fully capable of reflecting the instant cell volume variations remains stable, independent of the system used and application.

• Keywords
• Calcein, Cell volume changes, Fluorescence microscopy, Morphometry,
• MeSH
• Astrocytes cytology   physiology   MeSH
• 3T3 Cells MeSH
• Fibroblasts cytology   physiology   MeSH
• Fluorescent Dyes MeSH
• Microscopy, Fluorescence methods   MeSH
• Hypertonic Solutions MeSH
• Hypotonic Solutions MeSH
• Isotonic Solutions MeSH
• Microscopy, Confocal methods   MeSH
• Cells, Cultured MeSH
• Cerebral Cortex cytology   physiology   MeSH
• Mice MeSH
• Cell Size * MeSH
• Imaging, Three-Dimensional methods   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fluorescent Dyes MeSH
• Hypertonic Solutions MeSH
• Hypotonic Solutions MeSH
• Isotonic Solutions MeSH

• MeSH
• Hematocrit * MeSH
• Hepatectomy * MeSH
• Chromium Isotopes MeSH
• Blood Volume * MeSH
• Rats MeSH
• Laparotomy MeSH
• Body Weight MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chromium Isotopes MeSH

• Keywords
• CELL NUCLEUS *,
• MeSH
• Cell Nucleus * MeSH
• Humans MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

The dynamics of nuclear morphology changes during apoptosis remains poorly investigated and understood. Using 3D time-lapse confocal microscopy we performed a study of early-stage apoptotic nuclear morphological changes induced by etoposide in single living HepG2 cells. These observations provide a definitive evidence that nuclear apoptotic volume decrease (AVD) is occurring simultaneously with peripheral chromatin condensation (so called "apoptotic ring"). In order to describe quantitatively the dynamics of nuclear morphological changes in the early stage of apoptosis we suggest a general molecular kinetic model, which fits well the obtained experimental data in our study. Results of this work may clarify molecular mechanisms of nuclear morphology changes during apoptosis.

• Keywords
• 3D time lapsed confocal microscopy, Apoptosis, Apoptotic ring, Chromatin condensation, Kinetics, Live cell analysis, Mathematical modeling, Nuclear volume decrease,
• MeSH
• Single-Cell Analysis methods   MeSH
• Apoptosis physiology   MeSH
• Cell Nucleus physiology   ultrastructure   MeSH
• Hep G2 Cells MeSH
• Time-Lapse Imaging methods   MeSH
• Chromatin chemistry   metabolism   ultrastructure   MeSH
• Kinetics MeSH
• Microscopy, Confocal MeSH
• Humans MeSH
• DNA Packaging MeSH
• Models, Theoretical * MeSH
• Organelle Size physiology   MeSH
• Imaging, Three-Dimensional MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH

• MeSH
• Extracellular Space physiology   MeSH
• Ions MeSH
• Hydrogen-Ion Concentration MeSH
• Humans MeSH
• Microelectrodes MeSH
• Neurons cytology   physiology   MeSH
• Osmolar Concentration MeSH
• Receptors, Cell Surface physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Ions MeSH
• Receptors, Cell Surface MeSH

[K(+)](e) increase accompanies many pathological states in the CNS and evokes changes in astrocyte morphology and glial fibrillary acidic protein expression, leading to astrogliosis. Changes in the electrophysiological properties and volume regulation of astrocytes during the early stages of astrocytic activation were studied using the patch-clamp technique in spinal cords from 10-day-old rats after incubation in 50 mM K(+). In complex astrocytes, incubation in high K(+) caused depolarization, an input resistance increase, a decrease in membrane capacitance, and an increase in the current densities (CDs) of voltage-dependent K(+) and Na(+) currents. In passive astrocytes, the reversal potential shifted to more positive values and CDs decreased. No changes were observed in astrocyte precursors. Under hypotonic stress, astrocytes in spinal cords pre-exposed to high K(+) revealed a decreased K(+) accumulation around the cell membrane after a depolarizing prepulse, suggesting altered volume regulation. 3D confocal morphometry and the direct visualization of astrocytes in enhanced green fluorescent protein/glial fibrillary acidic protein mice showed a smaller degree of cell swelling in spinal cords pre-exposed to high K(+) compared to controls. We conclude that exposure to high K(+), an early event leading to astrogliosis, caused not only morphological changes in astrocytes but also changes in their membrane properties and cell volume regulation.

• MeSH
• Astrocytes physiology   MeSH
• Potassium pharmacokinetics   MeSH
• Potassium Channels, Voltage-Gated physiology   MeSH
• Glial Fibrillary Acidic Protein metabolism   MeSH
• Gliosis physiopathology   MeSH
• Hypotonic Solutions pharmacology   MeSH
• Immunohistochemistry MeSH
• Hydrogen-Ion Concentration MeSH
• Rats MeSH
• Membrane Potentials drug effects   physiology   MeSH
• Patch-Clamp Techniques MeSH
• Spinal Cord cytology   MeSH
• Osmotic Pressure MeSH
• Rats, Wistar MeSH
• Sodium metabolism   MeSH
• Sodium Channels physiology   MeSH
• Cell Size MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Potassium MeSH
• Potassium Channels, Voltage-Gated MeSH
• Glial Fibrillary Acidic Protein MeSH
• Hypotonic Solutions MeSH
• Sodium MeSH
• Sodium Channels MeSH

Volume transmission is a form of intercellular communication that does not require synapses; it is based on the diffusion of neuroactive substances across the brain extracellular space (ECS) and their binding to extrasynaptic high-affinity receptors on neurons or glia. Extracellular diffusion is restricted by the limited volume of the ECS, which is described by the ECS volume fraction α, and the presence of diffusion barriers, reflected by tortuosity λ, that are created, for example, by fine astrocytic processes or extracellular matrix (ECM) molecules. Organized astrocytic processes, ECM scaffolds or myelin sheets channel the extracellular diffusion so that it is facilitated in a certain direction, i.e. anisotropic. The diffusion properties of the ECS are profoundly influenced by various processes such as the swelling and morphological rebuilding of astrocytes during either transient or persisting physiological or pathological states, or the remodelling of the ECM in tumorous or epileptogenic tissue, during Alzheimer's disease, after enzymatic treatment or in transgenic animals. The changing diffusion properties of the ECM influence neuron-glia interaction, learning abilities, the extent of neuronal damage and even cell migration. From a clinical point of view, diffusion parameter changes occurring during pathological states could be important for diagnosis, drug delivery and treatment.

• Keywords
• astrocytes, diffusion, extracellular matrix, extracellular space, tortuosity, volume fraction,
• MeSH
• Anisotropy MeSH
• Astrocytes pathology   MeSH
• Diffusion MeSH
• Extracellular Matrix physiology   MeSH
• Humans MeSH
• Cell Communication physiology   MeSH
• Synaptic Transmission physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

In this study, we aimed to disclose the impact of amyloid-β toxicity and tau pathology on astrocyte swelling, their volume recovery and extracellular space (ECS) diffusion parameters, namely volume fraction (α) and tortuosity (λ), in a triple transgenic mouse model of Alzheimer's disease (3xTg-AD). Astrocyte volume changes, which reflect astrocyte ability to take up ions/neurotransmitters, were quantified during and after exposure to hypo-osmotic stress, or hyperkalemia in acute hippocampal slices, and were correlated with alterations in ECS diffusion parameters. Astrocyte volume and ECS diffusion parameters were monitored during physiological aging (controls) and during AD progression in 3-, 9-, 12- and 18-month-old mice. In the hippocampus of controls α gradually declined with age, while it remained unaffected in 3xTg-AD mice during the entire time course. Moreover, age-related increases in λ occurred much earlier in 3xTg-AD animals than in controls. In 3xTg-AD mice changes in α induced by hypo-osmotic stress or hyperkalemia were comparable to those observed in controls, however, AD progression affected α recovery following exposure to both. Compared to controls, a smaller astrocyte swelling was detected in 3xTg-AD mice only during hyperkalemia. Since we observed a large variance in astrocyte swelling/volume regulation, we divided them into high- (HRA) and low-responding astrocytes (LRA). In response to hyperkalemia, the incidence of LRA was higher in 3xTg-AD mice than in controls, which may also reflect compromised K+ and neurotransmitter uptake. Furthermore, we performed single-cell RT-qPCR to identify possible age-related alterations in astrocytic gene expression profiles. Already in 3-month-old 3xTg-AD mice, we detected a downregulation of genes affecting the ion/neurotransmitter uptake and cell volume regulation, namely genes of glutamate transporters, α2β2 subunit of Na+/K+-ATPase, connexin 30 or Kir4.1 channel. In conclusion, the aged hippocampus of 3xTg-AD mice displays an enlarged ECS volume fraction and an increased number of obstacles, which emerge earlier than in physiological aging. Both these changes may strongly affect intercellular communication and influence astrocyte ionic/neurotransmitter uptake, which becomes impaired during aging and this phenomenon is manifested earlier in 3xTg-AD mice. The increased incidence of astrocytes with limited ability to take up ions/neurotransmitters may further add to a cytotoxic environment.

• Keywords
• Alzheimer’s disease, ECS diffusion, astrocyte heterogeneity, astrocytes, ion uptake, volume changes,
• Publication type
• Journal Article MeSH

Activity-related changes in extracellular K+ concentration ([K+]e), pH (pHe) and extracellular volume were studied by means of ion-selective microelectrodes in the adult rat spinal cord in vivo and in neonatal rat spinal cords isolated from pups 3-14 days of age (P3-P14). Concomitantly with the ionic changes, the extracellular space (ECS) volume fraction (alpha), ECS tortuosity (lambda) and non-specific uptake (kappa'), three parameters affecting the diffusion of substances in nervous tissue, were studied in the rat spinal cord gray matter. In adult rats, repetitive electrical nerve stimulation (10-100 Hz) elicited increases in [K+]e of about 2.0-3.5 mM, followed by a post-stimulation K(+)-undershoot and triphasic alkaline-acid-alkaline changes in pHe with a dominating acid shift. The ECS volume in the adult rat occupies about 20% of the tissue, alpha = 0.20 +/- 0.003, lambda = 1.62 +/- 0.02 and kappa' = 4.6 +/- 0.4 x 10(-3) s-1 (n = 39). In contrast, in pups at P3-P6, the [K+]e increased by as much as 6.5 mM at a stimulation frequency of 10 Hz, i.e. K+ ceiling level was elevated, and there was a dominating alkaline shift. An increase in [K+]e as large as 1.3-2.5 mM accompanied by an alkaline shift was evoked by a single electrical stimulus. The K+ ceiling level and alkaline shifts decreased with age, while an acid shift, which was preceded by a small initial alkaline shift, appeared in the second postnatal week. In pups at P1-P2, the spinal cord was X-irradiated to block gliogenesis. The typical decrease in [K+]e ceiling level and the development of the acid shift in pHe at P10-P14 were blocked by X-irradiation. Concomitantly, continuous development of glial fibrillary acidic protein positive reaction was disrupted and densely stained astrocytes in gray matter at P10-P14 revealed astrogliosis. The alkaline, but not the acid, shift was blocked by Mg2+ and picrotoxin (10(-6) M). Acetazolamide enhanced the alkaline but blocked the acid shift. Furthermore, the acid shift was blocked, and the alkaline shift enhanced, by Ba2+, amiloride and SITS. Application of glutamate or gamma-aminobutyric acid evoked an alkaline shift in the pHe baseline at P3-P14 as well as after X-irradiation. The results suggest that the activity-related acid shifts in pHe are related to membrane transport processes in mature glia, while the alkaline shifts have a postsynaptic origin and are due to activation of ligand-gated ion channels.(ABSTRACT TRUNCATED AT 400 WORDS)

• MeSH
• Acetazolamide pharmacology   MeSH
• Amiloride pharmacology   MeSH
• Barium pharmacology   MeSH
• Magnesium Chloride pharmacology   MeSH
• Potassium metabolism   MeSH
• Electric Stimulation MeSH
• Extracellular Space physiology   MeSH
• gamma-Aminobutyric Acid pharmacology   MeSH
• Glutamates pharmacology   MeSH
• Homeostasis MeSH
• Hydrogen-Ion Concentration MeSH
• Rats MeSH
• Quaternary Ammonium Compounds pharmacology   MeSH
• 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid pharmacology   MeSH
• Glutamic Acid MeSH
• Cell Communication MeSH
• Spinal Cord growth & development   physiology   radiation effects   MeSH
• Neuroglia drug effects   physiology   MeSH
• Neurons drug effects   physiology   MeSH
• Rats, Wistar MeSH
• Aging physiology   MeSH
• In Vitro Techniques MeSH
• Hot Temperature MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Acetazolamide MeSH
• Amiloride MeSH
• Barium MeSH
• Magnesium Chloride MeSH
• Potassium MeSH
• gamma-Aminobutyric Acid MeSH
• Glutamates MeSH
• Quaternary Ammonium Compounds MeSH
• 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid MeSH
• Glutamic Acid MeSH
• tetramethylammonium MeSH Browser

Sunitinib is a broad-spectrum multitargeted tyrosine kinase inhibitor mainly used as second-line therapy for non-resectable gastrointestinal stromal or first-line treatment option of metastatic renal cell carcinoma (mRCC), and as an "off-label" option in pediatric oncology. It has been previously reported that sunitinib elevates the mean corpuscular volume of erythrocytes (MCV) in treated subjects. The aim of this study was to assess time-dependent changes of this effect and evaluate its possible clinical relevance. In this study, 179 adult and 21 pediatric patients with solid tumors treated with sunitinib were retrospectively analyzed. The laboratory and treatment-related data were collected for each treatment period. The regression model with a broken-line relationship was used to fit time dependence of the MCV. In the adult group, the MCV was increasing during the first 21.6 weeks (median) of treatment in a median level of 99.8 fL, where it stabilized. MCV increase was faster in the patients who suffered from treatment-related adverse events (21.3 vs. 24.6 weeks, p = 0.010). In the pediatric cohort, the MCV dynamics were similar to adults. In conclusion, MCV changes during sunitinib treatment in pediatric and adult patients may be of clinical utility in monitoring sunitinib treatment course.

• Keywords
• MCV, mean corpuscular volume, sunitinib, toxicity,
• MeSH
• Child MeSH
• Adult MeSH
• Erythrocyte Indices MeSH
• Indoles adverse effects   MeSH
• Carcinoma, Renal Cell * drug therapy   pathology   MeSH
• Humans MeSH
• Kidney Neoplasms * drug therapy   pathology   MeSH
• Antineoplastic Agents * adverse effects   MeSH
• Pyrroles adverse effects   MeSH
• Retrospective Studies MeSH
• Sunitinib pharmacology   therapeutic use   MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Indoles MeSH
• Antineoplastic Agents * MeSH
• Pyrroles MeSH
• Sunitinib MeSH