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Biomedical subjects

O Kempski

Publications and source records attributed to O Kempski.

At least 73 records · Page 4Linked to original sources
PubMed1996

Local cerebral blood flow in a rat cortical vein occlusion model.

The symptoms following sinus and vein occlusion observed in patients and experimental animals display a considerable variability that so far remains largely unexplained. In a rat cortical vein occlusion model using a photochemical thrombotic technique, we examined changes in the cerebral venous flow pattern by fluorescence angiography and regional cerebral blood flow (rCBF) and cerebral blood volume fraction (CBVF) by a modern laser Doppler "scanning" technique. Brain damage was assessed histologically. Fluorescence angiographic findings fell into two groups: group A, rats with an altered venous flow pattern after occlusion (n = 12), and group B, rats with interruption of blood flow and/or a growing venous thrombus (n = 5). In addition, sham-operated animals made up group C (n = 5). Extravasation of fluorescein, a massive decrease in rCBF, a short-lasting increase in CBVF, and regional brain damage were typical for group B. In addition, cortical CBF mapping revealed a transient hyperperfusion zone with hyperemia surrounding a hypoperfused ischemic core in group B. A circulation perturbation following venous occlusion appeared near those occluded cerebral veins without sufficient collateral flow. Furthermore, the venous thrombus continued to grow, accompanied by local critical ischemia and severe brain damage. Conversely, 71% of the animals (12 of 17) tolerated occlusion of a solitary vein without major flow disturbances or histological evidence of damage to the CNS (group A).

Animals↗
PubMed1996

Alterations of regional cerebral blood flow and oxygen saturation in a rat sinus-vein thrombosis model.

BACKGROUND AND PURPOSE: The pathophysiology of sinus-vein thrombosis (SVT) in patients and experimental animals is still poorly understood. This study was designed to examine and further elucidate the pathophysiological sequence of events, especially the relationship between local and regional blood flow and hemoglobin oxygen saturation (HbSO2) detected at identical locations. The use of both parameters as outcome indicators should be compared. METHODS: SVT was induced by ligation of the superior sagittal sinus (SSS) and slow injection of kaolin-cephalin suspension into the SSS in rats. Regional cerebral blood flow (rCBF) was assessed by laser-Doppler flowmetry together with regional HbSO2, which was measured by a microspectrophotometric technique at 48 identical locations for 90 minutes after SVT using a scanning technique. Fluorescence angiography was performed before and 30 and 90 minutes after SVT induction. After 48 hours the animals were killed for histology. RESULTS: The fluorescence angiographic findings could divide animals into three groups: (1) group A, with a solitary SSS thrombus (n=8); (2) group B, with a thrombosis of SSS and cortical veins (n=10); (3) group C, animals that had undergone sham operation (n=5). Decreases of rCBF and HbSO2 and brain damage were seen in group B but not in group A. The reduction of local HbSO2 preceded the flow decrease after sagittal sinus ligation but before thrombosis. Blood pressure in group A was found to be significantly higher after SVT than in groups B and C. CONCLUSIONS: The brain with acute extension of thrombus from the SSS into cortical veins experiences a critically reduced supply of blood and oxygen. CBF, local HbSO2, and repeated angiography can be helpful monitors for the early detection of critical conditions after SVT. Local HbSO2 has a greater sensitivity to predict outcome than lCBF. Moreover, therapies directed to improve perfusion pressure or reduce vascular resistance may open further therapeutic windows during SVT progression.

Animals↗
PubMed1996

Glioblastoma-associated circulating monocytes and the release of epidermal growth factor.

Monocytes/macrophages frequently infiltrate malignant gliomas and play a central role in the tumor-associated immune response as they process tumor antigen and present it to T-lymphocytes. Findings have accumulated that peripheral blood monocytes leaving the cerebral circulation become microglial cells and vice versa and that monocytes/macrophages may stimulate malignant tumor growth by some unknown mechanism. Most malignant gliomas express growth factor receptors, for example epidermal growth factor receptor (EGFR). The aim of this study was to determine whether peripheral blood monocytes of glioma patients release EGF, the appropriate ligand of gliomacell membrane-bound EGFR. Long-term cultured peripheral blood monocytes from 14 patients with malignant gliomas were compared to those from 12 controls (seven with nontumorous disease and five healthy individuals). Using an enzyme-linked immunosorbent assay for EGF, the EGF content of cell culture supernatants was determined at Days 7, 21, and 100 of culture. The EGF content (mean +/- standard error) of supernatants was 5.9 +/- 0.2 pg/ml/10(3) glioma monocytes versus 1.3 +/- 0.1 pg/ml/10(3) control monocytes at Day 7 of culture, 22.9 +/- 0.8 pg/ml/10(3) glioma monocytes versus 1.8 +/- 0.9 pg/ml/10(3) control monocytes at Day 21 of culture, and 23.4 +/- 0.7 pg/ml/10(3) glioma monocytes, and below detection levels for control monocytes at Day 100 of culture. Steroid treatment of glioma patients did not influence the EGF release of cultured monocytes. These data indicate that glioblastoma-associated peripheral blood monocytes may be distinct from those of healthy individuals. Moreover, this study indicates that subtypes of glioma-associated peripheral blood monocytes may support immunosuppression and promote growth of malignant glioma by releasing unusually high amounts of EGF.

Brain Neoplasms↗
PubMed1996

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 remained above control. There was no regulatory volume increase after cell shrinking in hypertonic media. Despite the severe anisotonic incubation, viability decreased only slightly without reaching statistical significance. In contrast to in vivo conditions, anoxia for 90 min with or without iodoacetate for additional inhibition of anaerobic energy metabolism neither caused neuronal cell swelling nor a decrease of viability. Reoxygenation after the anoxic period also did not induce volume and viability changes. Intracellular K+ of Neuro-2A cells was markedly decreased, while Na+ increased in a 1:1 ratio during complete energy failure by anoxia plus iodoacetate. A similar effect, occurring however somewhat delayed, was seen when the Neuro-2A suspension was exposed to iodoacetate alone. Anoxia without inhibition of glycolysis had no effect on intracellular ion concentrations, but lactate production was nearly six times higher than normal. In vitro, with a large extracellular volume and sufficient glucose supply, the energetic demands of Neuro-2A cells to maintain stable transmembraneous ion gradients during anoxia are obviously met by anaerobic glycolysis. The current results confirm that neuronal cells are able to adequately regulate cell volume in response to hyposmotic stress. On the other hand, maintenance of a normal cell size during complete energy deprivation suggests strongly that energy failure per se does not suffice to induce neuronal swelling. Cell swelling in cerebral ischemia in vivo thus appears a secondary phenomenon due to mediator mechanisms such as tissue acidosis or elevated extracellular glutamate levels.

Aerobiosis↗
PubMed1995

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 at 20 min, while 0.275 +/- 0.045 mM at 90 min. Addition of AA (0.1 mM) to cell-free medium was also associated with a steady decline of its concentration, although the decrease was markedly delayed as compared to the clearance in the presence of glial cells. AA was subjected to dose-dependent metabolisation in the cell suspension as demonstrated by the production of PGF2 alpha and LTB4. Following addition of 0.01 or 0.5 mM, concentrations of PGF2 alpha increased to a 1.9- or 4.9-fold level within 10 min, whereas those of LTB4 rose to a 1.3- or 33.7-fold level. This was attenuated or completely blocked, respectively, by the cyclo- and lipoxygenase inhibitor BW 755C. Formation of both metabolites from AA was also observed when studying astrocytes from primary culture. The current findings demonstrate an impressive efficacy of C6 glioma cells and astrocytes to clear arachidonic acid from the suspension medium and to convert the lipid compound into prostaglandins and leukotrienes. Uptake and metabolisation of AA by the glial elements may play an important role in vivo, for example in cerebral ischemia.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1994

Growth kinetics of a primary brain tissue necrosis from a focal lesion.

Secondary brain damage, such as brain edema or impairment of the cerebral microcirculation may evolve from tissue necrosis of the brain induced by trauma or ischemia. This laboratory has provided novel information on the secondary increase of a primary brain tissue necrosis resulting from a focal lesion. We have presently investigated more closely the growth kinetics of this process during 24 h after trauma. Rats were subjected to a standardized focal freezing injury of the brain. Area and volume of the resulting necrosis were quantitatively assessed by morphometry after different periods of survival (i.e., 5 min, 3, 6, 12, 18 and 24 h after trauma). The maximal area of necrosis increased by 45% (p < 0.001) during the posttraumatic observation period. Growth of necrosis after trauma was not limited to the early period, but continued between 12 and 24 h, amounting then to 29% (p < 0.05). The volume of necrosis calculated on the basis of histological serial sections was also observed to increase by 45%. The current findings confirm that a primary brain tissue lesion induced by a standard cryogenic injury, studied as model of a contusion focus in severe head injury, is subjected to secondary growth within a period of 24 h after trauma, longer periods of survival were not investigated yet. Quantification of lesion growth makes possible not only to study underlying mechanisms, but also of whether this process can be therapeutically inhibited.

Animals↗
PubMed1994

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 1.0 mM to 118.8 +/- 1.5% within 10 min. The swelling response to linoleic acid (18:2) was only about half of what was found when AA was administered at a concentration of 0.1 mM, whereas stearic acid (18:0) did not induce any cell volume changes. Inhibition of the cyclo- and lipoxygenase pathway by BW 755C did not prevent glial swelling from AA, whereas it was reduced by SOD, or almost completely abolished by the aminosteroid U-74389F, an antagonist of lipid peroxidation. Replacement of Na(+)- and Cl- -ions in the suspension medium by choline chloride was also associated with complete abolishment of cell swelling from AA. The results demonstrate an impressive efficacy of arachidonic acid to induce glial swelling which might be attributable to activation of lipid peroxidation by the fatty acid, leading to an increased Na(+)-permeability and subsequent influx of water into the cells.

Animals↗
PubMed1994

A nonlinear biomechanical model for evaluation of cerebrospinal fluid shunt systems.

In view of complications arising from physical properties of cerebrospinal fluid shunts, a biomechanical model of hydrocephalus was set up to study in vivo parameters that may influence their function. These include: intracranial pressure, compliance and pulses, intrathoracic, intra-abdominal, and subcutaneous pressures, and the effects of siphonage and repeated valve flushing. Each of these factors was studied separately upon shunt implantation in the model. Results of testing of a sample low-pressure valve with antisiphon device conformed with consumer information in regard to valve opening pressure and pressure flow measurements. No customer information, however, was supplied concerning the deleterious effects of direct subcutaneous pressure, variable degrees of siphonage, and repeated valve flushing that were demonstrated by the model. Such results indicate that shunts should be similarly tested prior to marketing and implantation in patients.

Biomechanical Phenomena↗
PubMed1994

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 non-malignant 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, pO2, pCO2 and temperature). After establishing constant baseline conditions, the cellular fluorescence intensity per cell volume, indicative of the uptake of Photofrin, and cell volume were assessed by flow cytometry, and cell viability was quantified by the trypan blue exclusion test. Photodynamic exposure of cells was performed using an argon-pumped dye laser system via a 600 microns optical fibre at energy density of 4 Joules at the cell surface (40 mW/cm2, 100 s). In comparison to endothelial and fibroblast cells, the melanoma cells exhibited no increased uptake of Photofrin, and no enhanced sensitivity to photodynamic therapy (PDT). However, the fluorescence intensity/volume of endothelial cells was two to three times higher at each concentration of the photosensitiser. Following PDT, reduction in cell viability was dependent on the concentration of Photofrin, and directly correlated with fluorescence intensity per cell volume. In addition, the cells of all three lines, treated by PDT, revealed dose-dependent changes in cell volume. Melanoma cells exhibited the most excessive increase. It is suggested that selective uptake of photosensitiser in vitro is not characteristic for tumour cells. The high uptake of Photofrin by endothelial cells may indicate that the vascular endothelium is a major target for PDT, leading to cessation of tumour blood flow and subsequent destruction of tumour tissue. In addition, PDT-induced swelling of tumour cells might represent and effect synergistically impairing tumour perfusion, and thereby promoting tumour death.

Animals↗
PubMed1994

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 mM. 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)carboxy-fluorescein. Administration of AA caused an immediate dose-dependent swelling of C6 glioma cells, even at a concentration of 0.01 mM. At this level cell volume increased within 20 min to 105.0% of control, at 0.1 mM to 111.0%, while at 1.0 mM to 123.7%. Following a phase of rapid cell volume increase, swelling leveled off during the subsequent observation period of 70 min. Viability of the C6 glioma cells was 90% under control conditions. It remained unchanged after raising AA concentrations to 0.1 mM. At 0.5 mM, however, cell viability fell to 72.8%, and at 1.0 mM to 32.7%. pHi of the glioma cells was 7.3 under control conditions. In parallel with the early swelling phase, AA led to a dose-dependent decrease of the intracellular pH and an elevated lactate production of the cells. During incubation with 0.1 mM AA, pHi decreased to 7.06 after 5 min, but recovered to normal subsequently. In addition, swelling-inducing properties of linoleic (18:2) or stearic (18:0) acid were analyzed for evaluation of the specificity of glial swelling induced by AA. Whereas stearic acid (0.1 mM) failed to induce a swelling response, linoleic acid (0.1 mM) was found to be effective. The volume increase of the glial cells, however, was only half of that found during exposure to AA at the same concentration. Further, glial swelling from AA or linoleic acid was completely inhibited by the aminosteroid U-74389F, an antagonist of lipid peroxidation. Finally, omission of Na+ ions in the suspension medium with replacement by choline led also to inhibition of the cell volume increase by AA. Experiments using astrocytes from primary culture confirmed the swelling-inducing properties of AA at a quantitative level, whereas vulnerability of the cells to AA was increased. The present results demonstrate an important role of AA in cytotoxic swelling and irreversible damage of glial cells at concentrations that occur in vivo in cerebral ischemia or trauma.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗
PubMed1994

Cerebral sinus and venous thrombosis in rats induces long-term deficits in brain function and morphology--evidence for a cytotoxic genesis.

The pathophysiology of cerebral venous infarctions is poorly understood, due partially to the lack of a suitable experimental model. Therefore, we developed a model in rats to study acute and long-term changes of brain function and morphology following thrombosis of the superior sagittal sinus. The superior sagittal sinus of rats was exposed, ligated, and injected with thrombogenic material. Thrombosis of the longitudinal sinus and ascending cortical veins was monitored by intravital fluorescence angiography. Histology was studied at 24 h and 4 weeks after thrombosis and changes in intracranial pressure, electroencephalogram (EEG), and tissue impedance were noted. Spontaneous locomotor activity was followed for 4 weeks after thrombosis. The effect of heparin treatment on tissue impedance was evaluated. Thrombosis of the superior sagittal sinus could be regularly induced, although pathological sequelae developed only if ascending veins were affected. Sinus and venous thrombosis was histologically characterized by bilateral, parasagittal infarctions. Thrombosis induction was followed by an increase in intracranial pressure from 4.7 +/- 1.6 to 12.8 +/- 2.4 mm Hg (n = 4) at 1 h after thrombosis, associated with an exponential rise in tissue impedance to 165 +/- 14% (n = 8) of the control. EEG changes were similar to those following global cerebral ischemia and remained pathological for up to 6 months after thrombosis (n = 6). As a permanent behavioral deficit spontaneous locomotor activity was reduced to 60 +/- 10% (n = 6) of the control. Finally, the administration of heparin (1 IU/g body weight) after thrombosis induction was found to reverse the pathological tissue impedance response of the brain. In conclusion, involvement of ascending cortical veins following sinus thrombosis appears to be critical for the development of irreversible tissue damage, such as infarction. Changes in intracranial pressure and tissue impedance suggest that the venous thrombosis was followed by brain edema of a predominantly cytotoxic nature. Venous thrombosis led to long-term changes of brain function, as demonstrated by persistent disturbances of the EEG or of the spontaneous locomoter drive. These deficits may be amenable to treatment with heparin.

Animals↗
PubMed1994

[Swelling and damage to nerves and glial cells by acidosis].

OBJECTIVE: Development of acidosis is a prominent pathophysiological factor in acute cerebral disorders, such as ischaemia or severe brain trauma. The impairment of the acid-base state in brain parenchyma among others is involved in the development of brain oedema, eventually leading to irreversible damage of neurons and glial cells. In the present study the pathophysiological role of acidosis for cytotoxic cell swelling and damage of glial and neuronal cells was investigated in vitro under conditions found in the ischaemic penumbra in vivo--the still viable perifocal border zone surrounding an infarct with elevated interstitial K(+)- and H(+)-concentrations. Assessment of cell swelling by acidosis was combined with experiments on underlying mechanisms as a basis for therapeutical interventions to inhibit cytotoxic brain oedema in vivo. METHODS: C6 glioma cells, astrocytes from primary culture, as well as Neuro-2A cells were cultivated, harvested and suspended as single cells under continuous control of pH, pO2, and temperature according to a standard procedure. Cell volume and cell viability were quantified by flow cytometry. Acidosis was induced by isotonic sulfuric- or lactic acid, respectively. RESULTS: Acidification of the medium led to cell swelling once pH fell below 7.0. Cell viability, however, was not affected by the increasing acidosis down to pH 6.2, while pH 5.6 or below was associated with cell death dependent on the duration of exposure. Acidosis-induced cell swelling was attenuated or completely inhibited by blocking of ion exchange mechanisms, such as the Na+/H(+)-antiporter, or elimination of Na+ ions from the medium. CONCLUSION: The present results provide new information on the nature of cytotoxic cell swelling and damage in central nervous system by acidosis under consideration of underlying mechanisms. Accordingly, acidosis-induced cell swelling is attributable to activation of ion exchange mechanisms, such as the Na+/H(+)- and Cl-/HCO3(-)-antiporter, in order to maintain a normal cellular acid-base state. This compensation process, however, is associated with the loss of cell volume control by net uptake of osmotic active solutes. Consequently, cell swelling occurring under these conditions is a result of regulatory mechanisms to defend homoeostasis rather than a consequence of cytotoxic cell damage. If cell swelling is inhibited by appropriate treatment, care should be exercised not to enhance the vulnerability of the nerve and glial cells.

Acid-Base Equilibrium↗
PubMed1994

Reduced mortality and brain damage after locomotor activity in gerbil forebrain ischemia.

BACKGROUND AND PURPOSE: Preischemic spontaneous locomotor activity was distinguished in this laboratory as a factor influencing outcome after 15 and 20 minutes of forebrain ischemia in gerbils. Histological investigations were carried out to analyze potential relations between postischemic survival and a reduction of cerebral damage by spontaneous locomotor activity. METHODS: Male Mongolian gerbils were divided into two groups, one with access to running wheels ("runners") and one kept in conventional cages ("nonrunners") for 2 weeks preceding forebrain ischemia of 15 or 20 minutes. A total of 99 gerbils were divided in subgroups and were allowed to recover for 2 weeks for assessment of survival. Other subgroups (n = 7 to 9) were killed at day 4 for quantitative histology of selectively vulnerable areas such as hippocampus, cortex, striatum, and thalamus. RESULTS: Two weeks after 15-minute ischemia, 44% of non-runners had survived compared with 90% of runners (P < .01). With 20-minute ischemia all runners survived compared with 21% of nonrunners. Quantitative histology (15-minute ischemia) revealed selective nerve cell injury in various cerebral regions in both groups. In runners, however, with the exception of the CA1 sector, damage was attenuated in cortex, striatum, and hippocampus. Furthermore, the extent of thalamic infarction was reduced (P < .05). CONCLUSIONS: Locomotor activity before global cerebral ischemia is highly efficient in protecting the brain as demonstrated by enhanced survival and a reduction of tissue damage in Mongolian gerbils. The mechanisms underlying this protection are currently unclear. However, further understanding of this intriguing phenomenon should enhance the understanding of ischemia pathophysiology and lead to the development of new treatment strategies.

Animals↗
PubMed1994

Enhanced interleukin-1 beta release and longevity of glioma-associated peripheral blood monocytes in vitro.

Interleukin-1 (IL-1) plays a controversial role in the immune response. Besides its activation of immune cells and juvenile central nervous system cells, monocyte-derived IL-1 may be able to stimulate the malignant transformation and proliferation of glial brain tumor cells expressing IL-1 receptors. The aim of this study was to determine the growth pattern and the IL-1 beta release of long-term cultured peripheral blood monocytes of glioma patients. At 6- to 7-day intervals, the vital monocytes, characterized by CD14 immunophenotyping, were counted. By the use of a specific IL-1 beta enzyme-linked immunosorbent assay, the IL-1 beta content of monocyte culture supernatants derived from 13 subjects with glioma and from 12 controls were compared at Days 7, 21, and 100 of culture. Cell clusters of monocytes derived from glioblastoma patients survived more than 250 days in culture, whereas control monocytes survived only up to 114 days. The IL-1 beta release of glioma-associated peripheral blood monocyte cultures was about 50 times higher as compared with control monocyte cultures. Dexamethasone treatment at the time of blood sampling and recurrences of the gliomas did not influence the increase in the IL-1 beta expression of glioma monocytes. It seemed that at least subsets of glioma-associated blood monocytes, although they had been removed from the circulation, remained activated for a long period of time. We conclude that increased IL-1 beta production of glioma-associated peripheral blood monocytes and their longevity in vitro may be features of aberrant immune cell subsets. In future studies, the exact phenotyping of monocyte subsets will be mandatory.

Adult↗
PubMed1993

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 pH 6.2 led to an increase in cell volume to 117.9 +/- 0.7% within 60 min. Torasemide (1 mM) inhibited the swelling response by 50% (P < 0.01). Cell swelling at pH 5.0, although more severe, was again attenuated by torasemide (P < 0.01). No effect was seen on the decrease in cell viability at this level of acidosis. Addition of glutamate led to a steady increase in cell volume which, contrary to cell swelling from lactacidosis, was not inhibited by torasemide. Inhibition of cell swelling from acidosis by this diuretic may be attributed to blocking of Cl-/HCO3- exchange mechanisms activated by acidosis. The lack of effect by torasemide in glial cell swelling from glutamate indicates operation of a different mechanism inducing cell swelling, for example cellular accumulation of the amino acid together with Na+ and water.

Acidosis, Lactic↗
PubMed1993

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 brain oedema. This flow threshold is in the neighbourhood of the ischaemic flow level causing irreversible tissue damage. Whereas in focal ischaemia oedema formation is a function of the severity of the flow decrease, it is a pathophysiological hallmark of early postischaemic recirculation in global cerebral ischaemia. Nevertheless, during complete interruption of cerebral blood flow translocation of interstitial fluid into the intracellular compartment occurs as manifestation of ischaemic cell swelling. Cell swelling under these conditions may, however, not necessarily indicate cell damage, but more likely a compensatory response attributable to the uptake of excitotoxic transmitters such as glutamate, and of K(+)-ions which are excessively released at the onset of ischaemia into the extracellular space. Purpose of the swelling process, thus, is clearance of extracellular fluid from this material to re-establish homeostasis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

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 concentration of Photofrin in the extracellular medium (r = 0.94; P < 0.001). Subsequent to increasing intervals of Photofrin exposure, cells were irradiated with laser light at 630 nm (40 mW/cm2, 4J). Cell viability as evaluated by trypan blue exclusion was significantly decreased with increasing concentrations of Photofrin in the medium, and significantly correlated with CFI during the plateau phase. After photodynamic treatment (PDT) cell fluorescence was reduced by about 15%. This was neither dose- nor time-dependent. On the basis of these findings we propose that CFI indicates photosensitizer uptake. This is also supported by the relation between CFI and phototoxicity. The latter also suggests that CFI might be useful to predict the PDT in vivo efficacy by this in vitro model. Besides measurements of photosensitizer uptake and cell photoxicity, the model demonstrates an excellent opportunity to study the molecular mechanisms of action associated with PDT.

Animals↗