Membrane damage thresholds for pulsed or continuous ultrasound in phagocytic cells loaded with contrast agent gas...

Cell membrane damage induced by pulsed or continuous ultrasound (US) activation of attached contrast agent gas bodies was examined in an in vitro model system. Monolayers of mouse macrophage-like cells were cultured on the inside of one window of an exposure chamber. The monolayers were incubated with Optison (Amersham Health Inc., Princeton, NJ) or Definity (Bristol-Myers Squib Medical Imaging, North Billerica, MA) and then rinsed to remove unattached gas bodies. A 3.5-MHz focused transducer was aimed at the chamber 3.7 cm away in a 37 degrees C water bath. The cells were scored for Trypan blue dye exclusion, with stained nuclei indicative of cell membrane damage. Exposure-response functions were approximated with exposure levels spaced 3-dB apart. Thresholds were located between the lowest exposure with statistically significant counts of blue-stained cells relative to sham exposures, and the next lower level. Thresholds with Optison included 0.05 MPa for 60-s continuous exposure duration, and 0.21 MPa for 0.6-micros pulses with 60-micros repetition period for 60-s pulsed exposure duration. Results were similar for Definity. Thresholds changed slowly with changes in timing parameters; for example, the threshold for a 0.6-micros continuous exposure (i.e., one pulse) was 0.84 MPa. Compared to 60-s exposure, this represents a factor of 16.8 increase in threshold for a factor of 10(8) decrease in exposure duration. The thresholds are less than the pressure amplitudes needed for nucleation of inertial cavitation, which suggests classification of the phenomenon as a form of gas body activation.