Effect of Torasemide — A New Cl--Transport Inhibitor on Glial Swelling by Lactacidosis and Glutamate

Cytotoxic brain edema, i.e. swelling of glial- and nerve cells is a common result of cerebral ischemia, brain trauma, metabolic disorders, and develops secondarily in vasogenic edema. During ischemia and severe head injury, brain tissue homeostasis is severely changed, and many parameters are simultaneously affected. To dissect and isolate the causal mechanisms leading to swelling of nerve- and glial cells, our laboratory has established an in vitro model [4, 5]. Thereby pathomechanisms can be studied in isolation without interference of epiphenomena taking place in damaged brain tissue under circumstances of cerebral ischemia or trauma. In previous studies the role of acidosis in cell swel…

Aufnahme und Phototoxizität von Hämatoporphyrinderivat in normalen und malignen Zellen

Die Photodynamische Therapie (PDT) stellt neben der Chemo-, Radio- und chirurgischen Therapie ein neues alternatives Therapiekonzept in der Behandlung von malignen Tumoren dar. Das Prinzip beruht auf der systemischen Applikation (i.v. Injektion) einer photosensibilisierenden Substanz, z.B. Hamatoporphyrinderivat (HPD), und der nachfolgenden Bestrahlung mit Licht geeigneter Wellenlange, z.B. Laserlicht (630 nm). Fur die tumorselektive Wirkung dieser Therapie werden die selektive Aufnahme bzw. vermehrte Speicherung der photoaktiven Substanzen im Tumorgewebe [1] sowie auch eine im Vergleich zum Normalgewebe erhohte Sensibuitat von Tumorgewebe gegenuber der PDT postuliert. Ungeklart ist, ob die…

Cytotoxic Glial Swelling by Arachidonic Acid

Arachidonic acid (AA, 20:4) is a major constituent of membrane phospholipids in brain tissue. Normally, the free fatty acid is present only in a small amount, but it accumulates under adverse conditions, such as ischemia or brain injury [1,9]. The release of free fatty acid involves activation of phospholipases and breakdown of membrane phospholipids. AA in particular is considered to mediate pathological processes. The polyunsaturated compound is a precursor of prostaglandins, leukotrienes, and oxygen-derived free radicals [11]. In cerebral ischemia concentrations of free AA of up to 0.5 mM/kg have been found in brain tissue [9].

An in vitro model to study cellular photosensitizer uptake and photodynamic dose-response relationships of tumor cells

Cellular fluorescence intensity (CFI) after incubation with varying concentrations of the photosensitizer Photofrin and the photodynamically induced dose-response relationships of hamster melanoma cells (A-MEL-3) were studied in a recently developed in vitro model. After administration of Photofrin to the extracellular serum-free medium, CFI was evaluated by flow cytometry together with constantly fluorescing latex particles used as a reference. After 5 min, 50% of maximal CFI was found, and after 60 min CFI was maximal. No further increase was obtained during the exposure to Photofrin over the incubation period of 4 h. During this plateau phase, CFI was significantly related to the concent…

Enhancing $l_i \to 3 l_j$ with the $Z^0$-penguin

Lepton flavor violation (LFV) has been observed in neutrino oscillations. For charged lepton FV decays only upper limits are known, but sizable branching ratios are expected in many neutrino mass models. High scale models, such as the classical supersymmetric seesaw, usually predict that decays $l_i \to 3 l_j$ are roughly a factor $\alpha$ maller than the corresponding decays $l_i \to l_j \gamma$. Here we demonstrate that the $Z^0$-penguin diagram can give an enhancement for decays $l_i \to 3 l_j$ in many extensions of the MSSM. We first discuss why the $Z^0$-penguin is not dominant in the MSSM with seesaw and show that much larger contributions from the $Z^0$-penguin are expected in genera…

Glial Swelling and Damage by Arachidonic Acid

Arachidonic acid (AA) is a major constituent of membrane phospholipids in brain tissue. Normally, the free fatty acid is present only in small amounts, but it accumulates under adverse conditions such as ischemia (Kinouchi et al. 1990). The release of free fatty acids involves activation of phospholipases and breakdown of membrane phospholipids. A A in particular is considered to mediate pathological processes. It is a polyunsaturated compound and precursor of prostaglandins, leukotrienes, and oxygen-derived free radicals (Wolfe 1982). In cerebral ischemia concentrations of free AA of up to 0.5mM/kg were found (Kinouchi et al. 1990). But also brain injury and seizures cause increased levels…

Chapter 6: Swelling of C6 glioma cells and astrocytes from glutamate, high K+ concentrations or acidosis

Publisher Summary With impaired energy supply, cell swelling results from the failure of Na+/K+-ATPase according to the pump-leak model of cell volume regulation. In a series of in vitro studies, glial cell volume changes during defined and strictly controlled alterations of the extracellular environment in vitro. Experiments were performed with C6 glioma cells and astrocytes from primary culture. The major advantages of using C6 cells are the rapid availability of large cell numbers necessary for reliable flow cytometric volume measurements, and the homogeneous cell size distribution permitting the detection of even subtle volume changes. The sodium-potassium pump was blocked by the cardia…

Anoxia in vitro does not induce neuronal swelling or death

To improve the understanding of neuronal cell swelling in cerebral ischemia, cell volume regulation, viability, intracellular electrolytes, and lactate production of Neuro-2A neuroblastoma cells were studied using an in vitro model. The volume regulatory capacity of Neuro-2A cells was assessed after incubation in hypo- and hypertonic media. Anoxia was studied alone and together with inhibition of glycolysis by iodoacetate. Reducing the tonicity of the incubation medium to 250, 200, or 150 mosm/l caused immediate swelling followed by a regulatory volume decrease within 20 min, which, however, was not complete. The final cell volume after regulation depended on the tonicity of the medium and …

Swelling, Intracellular Acidosis, and Damage of Glial Cells

Cerebral ischemia and severe head injury among others are associated with a limited availability of oxygen, leading to cell catabolism as well as anaerobic glycolysis. Resulting metabolites, such as arachidonic- and lactic acid, can be expected to leak into perifocal brain areas, contributing there to cytotoxic swelling and damage of neurons and glia. Since elucidation of mechanisms underlying cell swelling and damage in the brain is difficult in vivo, respective investigations were carried out in vitro using suspended glial cells. Thereby, effects of arachidonic acid (AA) and of lactacidosis on glial cell volume, intracellular pH (pHi), and cell damage were analyzed utilizing flow cytometr…

Swelling, Acidosis, and Irreversible Damage of Glial Cells from Exposure to Arachidonic Acid in vitro

Swelling and damage of C6 glioma cells and of primary cultured astrocytes were analyzed in vitro during incubation with arachidonic acid (AA; 20:4). The cells were suspended in a physiological medium supplemented with AA at concentrations of 0.001–1.0 m M. Cell swelling was quantified by flow cytometry with hydrodynamic focusing. Flow cytometry was also utilized for assessment of cell viability by exclusion of the fluorescent dye propidium iodide and for measurement of the intracellular pH (pHi) by 2′,7′-bis-(2-carboxyethyl)−5(and −6)carboxyfluorescein. Administration of AA caused an immediate dose-dependent swelling of C6 glioma cells, even at a concentration of 0.01 m M. At this level cel…

Relationship of Cerebral Blood Flow Disturbances with Brain Oedema Formation

Brain oedema is an important factor which compromises maintenance of the cerebral blood flow. Conversely, primary blood flow disturbances are leading to brain oedema. The mechanisms underlying blood flow impairment by brain oedema are associated with an increased regional tissue pressure in proportion to the degree of water accumulation in the parenchyma. The release of vasoactive mediator compounds might be considered in addition. Primary disturbances of the cerebral blood flow, such as focal or global cerebral ischaemia are leading to an increased cerebral water content. A decrease of the cerebral blood flow to ca. 40% of normal or below has been found to result in the development of brai…

Swelling of glial cells in lactacidosis and by glutamate: significance of Cl(-)-transport.

Swelling of glial and nerve cells is characteristic of brain damage in cerebral ischemia or trauma. The therapeutical efficiency of inhibition of Cl(-)-transport by a novel antagonist, the diuretic torasemide, on cytotoxic swelling of glial cells from lactacidosis, or glutamate was analyzed. Lactacidosis and the interstitial accumulation of glutamate are hallmarks of the pathophysiological alterations in ischemic or traumatic brain tissue. C6 glioma cells harvested from culture and suspended in a physiological medium were either exposed to pH 6.2, or 5.0 by lactic acid, or exposed to 1 mM glutamate at normal pH. Cell swelling and viability were quantified by flow cytometry. Lactacidosis of …

Relation of early Photofrin uptake to photodynamically induced phototoxicity and changes of cell volume in different cell lines.

For efficacy of photodynamic therapy, selective uptake and retention of photoactive substances has been postulated. Therefore, measurements were performed to find out whether the photosensitiser Photofrin® is taken up differently in malignant and non-malignant cells in vitro . In addition, the sensitivity of malignant cells and nonmalignant cells to photodynamic exposure was investigated, by quantifying viability and volume alterations of the cells. Bovine aortic endothelial cells, mouse fibroblasts and amelanotic hamster melanoma cells were suspended in a specially designed incubation chamber under controlled conditions (e.g. pH, p O 2 , p CO 2 and temperature). After establishing constant…

Mechanisms of Glial Swelling by Arachidonic Acid

The effect of arachidonic acid (AA, 20:4) was analyzed in vitro by employment of C6 glioma cells and astrocytes from primary culture. The cells were suspended in an incubation chamber under continuous control of pH, pO2, and temperature. Cell swelling was quantified by flow cytometry. After a control period, the suspension was added with AA at concentrations of 0.01 to 1.0 mM. Administration of AA induced an immediate, dose dependent swelling in C6 glioma cells or astrocytes. AA-concentrations of 0.01 mM led to an increase of the glial cell volume to 103.0 ± 1.0% of control, 0.1 mM to 110.0 ± 1.5%, and l.0 mM to 118.8 ± 1.5% within 10 min. The swelling response to linoleic acid (18 : 2) was…

Swelling and death of neuronal cells by lactic acid.

Lactacidosis occurring in cerebral ischemia or trauma is a major mechanism of cytotoxic brain edema and brain damage. Respective effects of lactacidosis were currently analyzed in vitro by employment of the murine neuronal cell line, Neuro-2A, in order to obtain a better understanding of specific mechanisms underlying cell swelling and cell death in comparison with glial cells. The cells were suspended in a physiological medium in the presence of lactic acid at increasing concentrations. Levels of acidosis reaching from pH 6.8-5.6 were obtained while other parameters, such as osmolarity and electrolyte concentrations, were maintained in the physiological range. Assessment of cell swelling a…

In-vitro Untersuchungen zu photodynamisch induzierten Dosis-Wirkungs-Beziehungen

Im Gegensatz zu fruheren Annahmen wurde von Rodgers 1990 in Frage gestellt, das aus der Quantifizierung der Fluoreszenz im Gewebe Aussagen uber die Konzentration des Photosensibilisators noch uber die zu erwartenden photodynami-schen Effekte moglich sind (1).

Glial ion transport and volume control.

K(+)-induced glial swelling results from an intricate interaction of transport and diffusion processes and metabolic stimulation, with many open questions remaining. Our concept of the major mechanisms involved can be summarized as follows: high extracellular K+ causes a burst-like stimulation of Na+/K+ ATPase and, hence, increases the metabolic demands. Lactate is produced; the cell is slightly acidified. To maintain a normal intracellular pH, the Na+/K+ antiporter extrudes protons and supplies Na+ for further Na+/K+ exchange. In addition, K+ ions enter the cell via membrane channels or furosemide-inhibitable transport. K+, Cl-, and lactate- ions accumulate as the osmotic basis for cell sw…

Enhancing $l_i 3 l_j$ with the $Z^0$-penguin