Membrane damage thresholds for 1- to 10-MHz pulsed ultrasound exposure of phagocytic cells loaded with contrast agent gas bodies in vitro

Abstract

Monolayers of mouse macrophage-like cells provide a model system for the study of bioeffects of pulsed ultrasound (US) activation of contrast agent gas bodies. In this study, the dependence of membrane damage on ultrasonic frequency was examined for gas bodies attached to the cells. The monolayers cultured on the inside of one window of an exposure chamber were incubated with 2% Optison (Amersham Health Inc., Princeton, NJ) and then rinsed to remove unattached gas bodies. The chamber was filled with culture medium plus 20% trypan blue stain solution and was mounted at the 3.8-cm focus of an US transducer in a 37 degrees C water bath. Transducers were used with center frequencies of 1.0, 2.25, 3.5, 5.0, 7.5 and 10.0 MHz. The 1-min pulsed exposures utilized two-cycle excitation with 1% duty cycle. After exposure, cells in the focal zone were scored for trypan blue dye exclusion, with stained nuclei indicative of cell membrane damage. Exposure-response functions were approximated by performing a series of exposures with peak rarefactional pressure amplitudes differing by a factor of radical 2 (i.e., 3 dB apart). Linear regressions were performed on selected data to determine a threshold pressure amplitude at each frequency. Thresholds ranged from 0.066 MPa at 1.0 MHz to 0.62 MPa at 10 MHz and were approximately proportional to the frequency. These thresholds are less than the pressure amplitudes needed for nucleation of inertial cavitation and have a different frequency dependence than the general Mechanical Index.