0000000000017736
AUTHOR
Alexander Baethmann
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…
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].
Cerebral protection against ischemia by locomotor activity in gerbils. Underlying mechanisms.
Background and Purpose A previous communication of this laboratory demonstrated reduced mortality and neuronal damage by spontaneous locomotor activity preceding forebrain ischemia in Mongolian gerbils. The present experiments seek to elucidate potential mechanisms of protection by measurement of cerebral blood flow, cerebral tissue conductance as an indicator of ischemic cell swelling, and the cerebral release of eicosanoids. Methods Gerbils were maintained either in conventional cages (nonrunners) or with free access to running wheels (runners) for 2 weeks preceding 15 minutes of forebrain ischemia. During ischemia and 2.5 hours of reperfusion, cerebral tissue conductance was determined …
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…
Glutamate Enhances Brain Damage from Ischemia and Trauma
The amino acid glutamate is a model agent to demonstrate the significance of neurotoxic mediator compounds in secondary brain damage from trauma, ischemia or other adverse conditions. Intensive research of the role of mediator compounds is clinically worthwile as more specific forms of treatment may emerge for the benefit of afflicted patients. In view of the great number of factors, cytokines, etc., which could play a role, it is mandatory that a mediator function in secondary brain damage is identified according to the stringent requirements established for that purpose. Glutamate has been shown — as is the case for only a few other agents — to meet all the criteria of a mediator of secon…
Electrical Impedance, ICP and Histology in Rats with Sagittal Sinus Occlusion
The diversity of clinical symptoms developing after thrombosis of the cerebral superior sagittal sinus is not well understood [2, 4], This is largely related to our ignorance of the underlying pathophysiology. An improvement in the pathophysiological understanding can be expected by experimental studies. Although animal models are available fot that purpose they are, however, in short supply. A few years ago, our laboratory in collaboration with the department of neurology at the University of Munich made another attempt to establish a reproducible experimental model for the induction of thrombosis of the superior sagittal sinus in rats. In the present studies this model has been used to ev…
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 …
Clearance and metabolism of arachidonic acid by C6 glioma cells and astrocytes.
Effects of increased levels of arachidonic acid (AA) were analyzed in vitro by employment of C6 glioma cells and astrocytes from primary culture. The cells were suspended in a physiological medium added with arachidonic acid (AA) in a concentration range from 0.01 to 0.5 mM. The concentration profiles of the fatty acid and AA-metabolites were subsequently followed for 90 min. AA was measured by gas chromatography, whereas the AA-metabolites PGF2 alpha and LTB4 by radioimmunoassay (RIA). Following administration of AA at 0.05 or 0.1 mM the medium was completely cleared from the fatty acid within 10 to 15 min. However, when 0.5 mM were added, AA concentrations of 0.36 +/- 0.055 mM were found …
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…
Mechanisms of arachidonic acid induced glial swelling
Accumulation of arachidonic acid (AA) in the brain during ischaemia may contribute to development of brain oedema. In this study we investigated the effect of selected drugs on AA-induced cytotoxic brain oedema in C6 glioma cells. Suspended C6 glioma cells were preincubated with drugs and AA (0.1 mM) was added. When no drug was administered cell volume increased immediately after the addition of AA with a maximum cell swelling of 13.1+/-1.9% at 15 min (mean +/- S.E. M.). Preincubation of cells with BW 755C, a dual inhibitor of cyclo- and lipoxygenases, showed no reduction in cell swelling from AA, whereas superoxide dismutase, amiloride and the protein kinase inhibitor H-9370 led to a signi…
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…