Dierk Suhr

Cavitation-generated free radicals during shock wave exposure: investigations with cell-free solutions and suspended cells

Extracorporeally generated shock waves as used in lithotripsy of urinary and biliary stones exhibit side effects in vivo. Furthermore, these shock waves destroy eukaryotic cells during in vitro treatment in suspension. A possible cause of these damaging effects might be cavitation, the growth and collapse of bubbles in liquids exposed to tensile stresses. During the collapse, temperature inside these cavitation bubbles rises up to several thousand K, leading to the formation of free radicals. We demonstrated the occurrence of cavitation-generated free radicals by direct reaction with fluorescent dyes in solution after shock wave treatment and investigated the resulting cell killing by variation of the cellular antioxidative defense status. We present evidence for the generation of intracellular free radicals during shock wave treatment of suspended cells.

Reduced cavitation-induced cellular damage by the antioxidative effect of vitamin E☆

Fragmentation of human urinary and biliary stones by shock waves in extracorporeal lithotripsy is accompanied by tissue damage. Both the fragmentation as well as the side effects are often attributed to cavitation. The hazardous potential of cavitation is not only of a physical nature but also of a chemical nature, because of the generation of free radicals, e.g. .OH, .H and .O2. After the application of shock waves, we have demonstrated cavitation-generated free radicals in cell-free solutions and also in the surviving and intact suspended MGH-U1 cells by hydroethidine measurements. Under electron microscopical inspection, the same cells exhibited perinuclear cisternae, damaged mitochondria and numerous intracellular vacuoles. The contribution of free radicals to cell damage was investigated by reducing the vitamin E level in rats by a tocopherol free diet and by incubating L1210 cells in a tocopherol enriched medium. After 250 shock waves, ex vivo erythrocytes revealed a 75% increase in total cell disruption over cells from non-depleted rats. The in vitro experiments with L1210 cells exhibited a moderate protection by the addition of this scavenger of free radicals.

Disturbance of cellular calcium homeostasis by in vitro application of shock waves

Extracorporeally generated shock waves used in lithotripsy of urinary and biliary stones exhibit tissue lesions in vivo and destroy or damage cells in vitro. The involvement of cavitation-generated free radicals in these harmful effects is discussed controversially. We investigated changes in cytoplasmic calcium concentration and intracellular calcium localization after shock-wave treatment of suspended cell cultures using flow cytometry and electron microscopy and present evidence for the disturbance of mitochondrial Ca2+ a sequestration and, therefore, for a chemically induced cell injury.

Arachidonic acid reversibly reduces gap-junctional permeability

We investigated the effect of arachidonic acid on the intercellular communication of a rat mammary tumor cell line. This polyunsaturated fatty acid reversibly reduced the intracellular pH and raised the concentration of cytoplasmic free Ca 2+ . However, neither low pH nor elevated [Ca 2+ ] j influenced gapjunctional coupling. Measurements with the double whole cell patch-clamp technique imply that arachidonic acid influences gap-junctional permeability via a direct lipid-protein interaction.

Bioeffects of diagnostic ultrasound in vitro

Biological effects induced by ultrasound were frequently reported for continuous wave (cw) mode. Thresholds for the onset of bioeffects of pulsed ultrasound, starting from diagnostic conditions, have not yet been defined by standardized in vitro models. We therefore investigated the effects of pulsed ultrasound on cultured cells using diagnostic ultrasound devices, a selfmade transducer and a sonochemical laboratory reactor tunable from pulsed diagnostic conditions to cw ultrasound. Additionally, we determined physical parameters of the ultrasonic field by different types of hydrophones. Sonochemical reactions and the effects induced by the ultrasonic fields in cultured cells indicated a threshold for bioeffects.

Are biological effects of shock waves caused by free radicals

Stone fragmentation in extracorporeal shock wave lithotripsy as well as accompanying tissue damage are attributed to cavitation. We attempted to demonstrate and localize the occurrence of cavitation in cell-free solutions and MGH-U1 cells using sensitive dyes for the detection of cavitation-generated free radicals.

In-vitro-Testsysteme zur Untersuchung der Wirkung von Ultraschallpulsen

Zusammenfassung Die Untersuchungen einiger diagnostischer Ultraschallgerate, einschlieslich farbkodierter Puls-Doppler-Gerate, mit In-vitro-Testsystemen zeigten keine auf Kavitation beruhenden Effekte. Nach der Beschallung von suspendierten Erythrozyten war weder deren Zerstorung noch eine Freisetzung von Hamoglobin nachweisbar. Durchfluszytometrische Untersuchungen von beschallten Maus-Leukamie-Zellen ergaben keine signifikanten Veranderung der Zellzahl und der Vitalitat der Zellen. Auch in zellfreien Experimenten war keine sonochemische Aktivitat in Farbstofflosungen nachzuweisen. So erzeugte die Beschallung des Farbstoffes Dichlorofluorescin keine Steigerung des Oxidationsproduktes Dichlorofluorescein. Wachstumskurven beschallter Zellen sowie licht- und elektronenmikroskopische Histologie erbrachten keine Veranderungen gegenuber den Kontrollgruppen. Eine biologische Ultraschallwirkung in vitro konnte mit keinem der untersuchten diagnostischen Gerate festgestellt werden. Bei den anschliesend durchgefuhrten Untersuchungen mit einer Hochgeschwindigkeitskamera wurden unter diesen Bedingungen keine Kavitationsereignisse beobachtet. In weitergehenden Experimenten mit einem experimentellen Labor-Ultraschallreaktor (Schwellenwerte Freifeldbedingungen: 3 ms Pulslange, Pulswiederholrate 200 Hz, Nahfeldbedingungen: 20 µs, Pulswiederholrate: 5 kHz) konnte das Einsetzen sonochemischer Reaktionen und Bioeffekte beobachtet werden.