Thomas Bräuner

Comparative measurements of membrane potentials with microelectrodes and voltage-sensitive dyes

The usefulness of a new voltage-sensitive fluorescent dye, the membrane permeant negatively charged oxonol dye diBA-C4-(3)-, was evaluated by measuring the membrane potentials of BICR/M1R-k and L cells with glass microelectrodes and simultaneously recording the fluorescence of the stained cells. The membrane potential of BICR/M1R-k cells was varied between -25 mV and -90 mV by changing the bicarbonate concentration in the medium or by voltage clamping. To avoid any interference by the inserted electrodes with the fluorescence measurement of the cytoplasm, the cells were fused by polyethyleneglycol to form giant cells (homokaryons). These homokaryons also allowed penetration by two glass microelectrodes without causing a serious leakage of the plasma membrane. The slow responding dye diBA-C4-(3)- had a fluorescence response of about 1% per mV. Mathematical analysis of the fluorescence changes after voltage clamping revealed a first-order reaction with a rate constant between 0.1 min-1 and 0.8 min-1, depending on the cell size which was determined by the number of nuclei per homokaryon. A model for the mechanism of the fluorescence changes is proposed.

Biological effects of shock waves

SummaryExtracorporeal shock wave lithotripsy has become established worldwide as the method of choice for the treatment of nephrolithiasis and ureterolithiasis over the last 10 years. Although initial studies showed no damaging effects of the shock waves on organs and tissues, numerous recent reports have presented evidence for severe acute effects and chronic complications after shock wave treatment. The pathophysiological effects on kidneys and the histopathological effects on organs or tissues in man and animal, and also the effects on cells in culture and tumors are sumarized. Suspended and immobilized cell cultures were used to characterize and quantify the efficacy of shock wave. Extended applications of shock waves and possible modifications to shock wave generators are discussed.

Effect of shock waves on suspended and immobilized L1210 cells

L1210 mouse leukemia cells have been exposed to different doses of shock waves generated by underwater spark discharge at 18 kV in an experimental lithotripter (XL1, Dornier). Histological and flow cytometric investigations revealed severe damage and a LD50 of about 420 shock waves when the cells were treated as suspensions. Cells immobilized in gelatine, however, were unaffected, indicating that secondary effects are responsible for the cellular damage. Possible mechanisms such as cavitation, jets, and shear forces are discussed.

Histopathology of shock wave treated tumor cell suspensions and multicell tumor spheroids

L1210 mouse leukemia cell suspensions exposed to 500 shock waves (SW) in an experimental lithotripter (XL1, Dornier) revealed severe cellular damage. Apart from cell fragments and cellular debris, cells exhibited alterations of shape, vacuolization of the cytoplasm, perinuclear cisternae, swelling of mitochondria or rupture of the mitochondrial fine structure, and permeabilization of the cell membrane. Treatment of multicell tumor spheroids of both HeLa and EMT6/Ro cells in suspension with 500 SW resulted either in loss of peripheral cells and serious cellular damage in the outer regions or in a fragmentation of the spheroids. Many of the geometrically intact cells exhibited the same histopathological alterations as the suspended L1210 cells. Immobilization of the spheroids in agar or gelatine, however, prevented spheroids from being agitated and accelerated during SW-exposure. After treatment with 500 SW, spheroids immobilized in gelatine were not different from control cultures, as investigated with light- and electronmicroscopy. From our results we conclude that spheroids in suspension are subject to cavitation and liquid jet formation, causing not only acceleration and shearing forces but also collisions which account for the observed cell damage.

Zell- und Gewebsveränderungen durch eine Stosswellenbehandlung

Die extrakorporale Stoswellenlithotripsie kann seit Beginn ihrer klinischen Anwendung im Jahre 1980 bedeutende Erfolge bei der beruhrungslosen Nlerensteinzertrummerung vorweisen, was dazu fuhrte, das sie mittlerweile auch fur die Behandlung von Gallensteinen angewendet wird. Da offensichtlich wesentlich geringere Nebenwirkungen als nach Operationen auftreten, hat sich diese Methode weltweit rasch ausgebreitet und es ist nicht verwunderlich, wenn nunmehr auch uber eine Anwendungserweiterung in Richtung Tumortherapie spekuliert wird. Dazu mussen allerdings die zellularen Auswirkungen der Stoswellenbehandlung von Geweben genauer charakterisiert werden.

A million cells in search for contact : multicell spheroids not only for cancer research

Three-dimensional, spherical aggregates of cells - so-called multicell spheroids - have many practical applications. In cancer research, for example, they contribute to a drastic reduction in the number of experiments with animals. The authors of the following article were awarded a special DM 10,000 prize under the Felix-Wankel-Tierschutz-Forschungspreis in November 1986 for their work on intercellular communication in multicell spheroids.