The role of pulse repetition frequency in ultrasonic contrast agent stability and cell death

Ultrasound exposure of phagocytic cells with contrast agent microbubbles attached can cause cell death. The influence of pulse repetition frequency (PRF) on the occurrence of such cell death was studied. Phagocytic cells of mouse macrophage cell line RAW-264.7 were grown as monolayers on thin Mylar sheets by standard culture methods. These cells retain contrast agent when incubated with 5% Optison^® (Amersham Health, Princeton NJ) for 15 minutes and then rinsed to remove unattached microbubbles. A sealed chamber was formed with a second thin sheet placed 1 mm from the first sheet containing cells. Focused ultrasound exposures were implemented at a frequency of 2.25 MHz with 46 cycle pulses and PRF´s of 1 kHz, 100 Hz, 10 Hz, 1 Hz, and 0.1 Hz. All ultrasound exposures were performed with a peak rarefactional pressure of 2 MPa in a degassed water bath. A 1 MHz receiver transducer measured the scattered signal from the monolayer containing cells and contrast agent. The signals were captured using a high speed digitizing board and range gated from within the chamber. The frequency spectrum was then computed and normalized to a control spectrum from linear scatterers (Zerdine^TM, CIRS, Norfolk, VA). Photomicrographs of the cell monolayer were made in the ultrasound beam area before and after exposure, and a dye exclusion test (Trypan Blue) was used to find the percentage of blue stained cells indicating loss of cell membrane integrity and cell death. Cell death (counted as both the absent and stained cells) was examined and related to acoustic emission. Acoustic scattering power was higher for Pulse #1 than that for Pulse #2. For 5% Optison^®, cell death at 1 kHz PRF was lower that that for 100 Hz, 10 Hz, 1 Hz, 0.1 Hz.