Acoustic energy determines haemoglobin release from erythrocytes by extracorporeal shock waves in vitro.

Haemoglobin release from erythrocytes by extracorporeal shock waves from an electrohydraulic lithotripter was quantified and correlated with the acoustic energy administered to the cell container. Cells were exposed in 2-, 5.9-, and 10.5-mL vials to 100 shock waves delivered at a low, medium and high lithotripter output setting, both with and without covering of the central ellipsoidal axis by a metal cage. Using the identical set-up, previous experiments had shown that the fragmentation efficiency was linearly correlated with the delivered acoustic energy. As a result, shock waves generated from 0.83 microgram mJ-1 (in 2-mL vials) to 1.53 micrograms mJ-1 (in 10.5-mL vials) haemoglobin. At all vial types, the amount of haemoglobin correlated linearly with the delivered acoustic energy (r = 0.96 in 2-mL, r = 0.97 in 5.9-mL and r = 0.98 in 10.5-mL vials). It was independent of the presence of the cage.

Effect of shock waves and cisplatin on cisplatin-sensitive and -resistant rodent tumors in vivo.

The effect of a combination of shock waves with cisplatin was examined in vivo with subcutaneously implanted amelanotic melanomas (A-Mel 3) in Syrian golden hamsters and cisplatin-sensitive or cisplatin-resistant fibrosarcoma (SSK2/0 and SSK2/R2) in C3H mice. In all 3 tumor models, 4 treatment modalities were compared: control, cisplatin treatment, shock waves and the combination of shock waves and cisplatin. Shock waves significantly delayed tumor growth in all 3 tumor models when compared to the respective control group. Cisplatin alone delayed the growth of A-Mel 3 and SSK2/0, whereas SSK2/R2 remained uninfluenced by the drug. In all 3 tumor models the combined treatment with shock waves and cisplatin additively and significantly delayed tumor growth. In A-Mel-3-bearing animals the combined treatment significantly increased survival time. The growth of SSK2/0 and SSK2/R2 tumors was delayed to a similar extent by the combined treatment modality as compared to shock-wave treatment alone. This indicates that the cisplatin resistance of SSK2/R2 tumors has been overcome by the simultaneous shock wave treatment. An increased intracellular cisplatin accumulation in the tumors due to shock wave exposure is suggested as the mechanism of interaction between shock waves and cisplatin.

Permeabilization of the plasma membrane of L1210 mouse leukemia cells using lithotripter shock waves.

Permeabilization of L1210 cells by lithotripter shock waves in vitro was monitored by evaluating the accumulation of fluorescein-labeled dextrans with a relative molecular mass ranging from 3,900-2,000,000. Incubation with labeled dextran alone caused a dose- and time-dependent increase in cellular fluorescence as determined by flow cytometry, with a vesicular distribution pattern in the cells consistent with endocytotic uptake. Shock wave exposure prior to incubation with labeled dextran revealed similar fluorescence intensities compared to incubation with labeled dextran alone. When cells were exposed to shock waves in the presence of labeled dextran, mean cellular fluorescence was further increased, indicating additional internalization of the probe. Confocal laser scanning microscopy confirmed intracellular fluorescence of labeled dextran with a diffuse distribution pattern. Fluorescence-activated cell sorting with subsequent determination of proliferation revealed that permeabilized cells were viable and able to proliferate. Permeabilization of the membrane of L1210 cells by shock waves in vitro allowed loading of dextrans with a relative molecular mass up to 2,000,000. Permeabilization of tumor cells by shock waves provides a useful tool for introducing molecules into cells which might be of interest for drug targeting in tumor therapy in vivo.

Destruction of gallstones and model stones by extracorporeal shock waves.

The disintegration effectivity of an electrohydraulic lithotripter was evaluated by determining the acoustic energy that had to be applied, until all fragments of three artificial materials and human gallstones were cleared from a basket of 2.8 mm mesh size. The lithotripter had either an open ellipsoid, or the ellipsoidal axis was covered with a metal cage as used in clinical lithotripters to house the ultrasound scanner. Fragmentation was assessed at a low, medium and high voltage setting using 9 and 16 mm breeze block marbles, considered to be primarily fragmented by a cavitation-mediated mechanism; 16 mm glass marbles, considered to be primarily fragmented by a direct shock wave effect; 12 and 15 mm plaster balls as commonly used to monitor lithotripter output; and gallstones with a mean diameter of 16 mm. As a result, the acoustic energy for the disintegration of 9 and 16 mm breeze block marbles was 620 and 670 mJ cm-3, of glass marbles 3369 mJ cm-3, of 12 and 15 mm plaster balls 1599 and 1764 mJ cm-3 and of gallstones 8050 mJ cm-3. It was largely independent of pulse energy, specimen size and configuration of the shock wave source. It is concluded that acoustic energy is a major determinant of disintegration, independent of the presumed mechanism of destruction.

Extracorporeal shock waves stimulate frog sciatic nerves indirectly via a cavitation-mediated mechanism.

Shock waves (SWs) are single pressure pulses with amplitudes up to over 100 MPa, a rise time of only a few nanoseconds, and a short duration of approximately 2 microseconds. Their clinical application for stone destruction causes pain, indicating nerve stimulation by SWs. To examine this phenomenon, sciatic nerves of frogs were exposed to SWs in an organ bath. The SWs were generated with an experimental Dornier lithotripter model XL1 at an operating voltage of 15 kV. The nerves were mounted in a chamber which allowed electrical nerve stimulation and the registration of electrically and SW-induced compound action potentials (SWCAPs). The chamber was filled with frog Ringer's solution. In a standardized protocol. The first experiment established that 95.0 +/- 4.7% of administered SWs induced action potentials which were lower in amplitude (1.45 +/- 1.14 versus 1.95 +/- 0.95 mV, p = 0.004) but similar in shape to electrically induced compound action potentials. In a second experiment, it was shown that the site of origin of the SWCAPs could be correctly determined by simultaneous recording of action potentials at both ends of the nerve. The mechanism of shock wave stimulation was examined by experiments 3 and 4. In experiment 3, in contrast to the previous experiments, SW exposure of the nerves was performed 6 cm outside the shock wave focus. This resulted in a mean probability of inducing a SWCAP of only 4%. After gas bubble administration, this probability increased to 86% for the first SW released immediately after bubble application and declined to 56% for the second, 21% for the third, to 0 for the 10th SW after fluid injection. This indicates that cavitation, the interaction between shock waves and gas bubbles in fluid or tissues, was involved in SWCAP generation. In experiment 4, nerves were again exposed in the focus, however, the Ringer's solution surrounding the nerve was replaced by polyvinyl alcohol (PVA). PVA is a solution with low cavitation activity.In PVA, the excitability was markedly diminished to 11.0 +/- 5.1% compared with 96.0 +/- 4.4% in control nerves exposed in Ringer's solution. In conclusion, bioeffects of SWs on nervous tissue appear to result from cavitation. It is suggested that cavitation is also the underlying mechanism of SW-related pain during extracorporeal SW lithotripsy in clinical medicine.

In vitro interaction of lithotripter shock waves and cytotoxic drugs.

The effect of a combination of lithotripter shock waves and cytotoxic drugs was examined in vitro. L1210 cells in suspension were exposed to shock waves during incubation with cislatin, doxorubicin, daunorubicin, THP-doxorubicin, or aclacinomycin. Proliferation was determined using the 3-4,5 dimethylthiazol-2,5 diphenyl tetrazolium bromide assay. Dose enhancement ratios were calculated for each drug in order to determine the effect of the additional exposure to shock waves. In addition, partition coefficients and IC50s of the drugs were determined. It was found, that the dose enhancement ratios increased for the drugs with decreasing cytotoxicity. The effect of all five drugs was enhanced by shock waves to a higher degree at 7 min incubation as compared to 50 min incubation. The effect of cisplatin was most significantly enhanced, with a dose enhancement ratio of 6.7 at 7 min incubation. The enhancement increased with the operating voltage used for generating the shock waves, and was only present when cells were exposed to shock waves during the incubation with the drug. An increase in cellular membrane permeability is proposed as the mechanism of interaction between shock waves and drugs.

Sonographic imaging of extracorporeal shock wave effects in the liver and gallbladder of dogs.

During extracorporeal shock wave lithotripsy, changes in tissue echogenicity are observed by ultrasound. Their significance is not known. An experiment was performed in which 3,000 extracorporeal shock waves were applied under sonographic observation to the gallbladder wall of 6 dogs. No stones had been implanted, but transient shadows appeared simulating jumping stone fragments in the bladder. Echoes within the bladder lumen occurred in 3 dogs and were associated with hemorrhage into the lumen; in an additional ex vivo experiment, shock waves generated echoes in bile only after injection of a small amount of blood. In the liver, a transient increase in echogenicity was noted after a few shocks; it coincided with the region of tissue damage. Intense focal echoes occurred in the liver of 2 dogs at sites where a hematoma was found at autopsy. It is concluded that an increased focal echogenicity is an indicator of tissue damage by shock waves. The sonographic changes are thought to be caused by the transient generation of gas bubbles. The interaction of shock waves with gas bubbles is an established powerful mechanism which could explain the generation of tissue damage by shock waves.

Influence of dissolved and free gases on iodine release and cell killing by shock waves in vitro.

Exposure of a potassium iodide solution to lithotripter shock waves resulted in formation of iodine with the amount of iodine depending on the gas dissolved in the solution. Iodine yield was higher with O2 and Ar, as compared to CO2 and N2O; degassed solution revealed the lowest iodine yield. Exposure of L1210 mouse leukemia cells to shock waves reduced the number of viable cells with no difference between O2-, Ar-, or N2O-equilibrated and degassed conditions. CO2 equilibration resulted in a more pronounced reduction. The difference between chemical and biological effects argues against the involvement of free radicals in cell killing by shock waves. In additional experiments, gas bubbles of various sizes were introduced into the test vials. Addition of a 10 microL gas bubble revealed an over 10-fold increase in iodine yield from degassed potassium iodide solution with all gases. Addition of a gas bubble also reduced the number of viable cells again with no difference between the gases. It is suggested that shock wave-gas bubble interaction is an important mediator of iodine release and cell killing by shock waves.

Biological effects of shock waves: cell disruption, viability, and proliferation of L1210 cells exposed to shock waves in vitro.

L1210 cells were exposed in suspension to shock waves generated with a Dornier XL1 lithotripter. After 1000 discharges at 25 kV, the number of nondisrupted cells was 15% and the number of trypan blue excluding cells was 7% as compared to 100% in sham treated controls; the shock-wave effect was more prominent at higher voltages and less prominent at higher discharge numbers when compared at similar electrical input energies. Overall proliferation of cells which were trypan blue negative after exposure exceeded 70% of the proliferation of sham treated controls, except after 1000 shocks at 25 kV, where proliferation was reduced to 42%. The latter reduction in proliferation was found to be due to a reduced growth for 24 h after exposure, with a return to normal proliferation during the following days. Limiting dilution analysis revealed that the reduced growth was mainly due to a transitory increase of the doubling time and not to a reduction of the number of proliferating cells. Cell disruption by shock waves was completely inhibited by exposing the cells at an elevated pressure of 101 atmospheres, pointing to the possible involvement of cavitation in the shock wave effect.

Influence of the shock wave application mode on the growth of A-Mel 3 and SSK2 tumors in vivo.

We examined the influence of different shock wave application modes with a Dornier XL1 electrohydraulic lithotripter on the growth of A-Mel 3 and SSK2 tumors implanted under the dorsal skin of hamsters or mice. In a basic protocol, 500 shock waves a day on 4 consecutive days were administered at a discharge rate of 100 waves per minute and focused to the tumor center. This did not affect A-Mel 3 growth. A similar result was obtained with the basic protocol modified to 1000 shock waves a day and a wave application rate of 100 shock waves per second. Growth of A-Mel 3 and SSK2 tumors was significantly delayed, when the basic protocol was used, but the 500 shock waves a day were distributed over four points at the tumor edges and the tumor center. With the same shock wave protocol, lowering the water level over the tumor from 10 cm to 1 cm induced temporary regressions of SSK2 tumors. This was not due to the higher energy applied to the tumor, since twice the number of shock waves (1000 a day instead of 500 a day) was applied at a high water level and did not induce regressions. Four consecutive treatments with intervals between treatments shortened to 3 h and an additional treatment 12 h later at a low water level completely controlled tumor growth in 8 out of 12 SSK2 tumors for more than 150 days. The result showed that addition of a reflected wave from the water surface was most important for the shock wave effect, and suggested that shock wave devices generating similar wave forms should be applied for tumor therapy.

In vitro cytotoxic activity of lithotripter shock waves combined with adriamycin or with cisplatin on L1210 mouse leukemia cells.

The effect of a combined treatment with shock waves generated by a lithotripter and Adriamycin or cisplatin was examined in cells that acutely survived exposure to shock waves and proliferated afterwards. Batches of 2 x 10(6) cells were exposed to the respective drug for 50 min or for 50 min plus 72 h. During the 50-min drug exposure 500 shock waves were applied at 25 kV. The growth as a percentage of the control was determined after 72 h by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cells treated with shock waves alone showed a growth inhibition as compared to control cells. For a 50-min drug exposure with Adriamycin the dose enhancement ratio did not exceed 1.3. For a 50-min drug exposure with cisplatin at concentrations of 0.5 micrograms/ml and 5.0 micrograms/ml, growth (as a percentage of the control) after combined treatment was significantly reduced as compared to cisplatin treatment alone; the dose enhancement ratio was 3.2 at 50% growth compared to the control. This indicates that shock waves can increase the susceptibility of L1210 cells to cisplatin. For a 50-min plus 72-h drug exposure no effect of an additional treatment with shock waves, as compared to chemotherapy alone, could be observed.