In vivo microscopy of targeted vessel occlusion employing acoustic droplet vaporization.

OBJECTIVE: Embolotherapy is a potential means to treat a variety of cancers. Our approach-gas embolotherapy-introduces the droplets upstream from the tumor and then acoustically activates them to form bubbles for occlusion-a process known as ADV. We wanted to provide the first optical documentation of ADV, lodged bubbles, or vessel occlusion in vivo. METHODS: We used the rat cremaster muscle for in vivo microscopy. Perfluorocarbon droplets were administered into the aortic arch. Ultrasound exposures in the cremaster induced vaporization. The cremaster was examined pre- and post-exposure for ADV-related effects. Two sets of experiments compared the effect of exposure in the capillaries versus the first order arteriole. RESULTS: Bubbles that lodge following capillary exposure are significantly larger (76 µm mean length, 36 µm mean diameter) than those following feeder vessel exposure (25 µm mean length, 11 µm mean diameter). Despite the differing sizes in bubbles, the ratio of bubble length to the hydraulic diameter of all lodged bubbles was 2.11 (±0.65; n = 112), which agrees with theoretical predictions and experimental observations. CONCLUSIONS: Our results provide the first optical evidence of targeted vessel occlusion through ADV. These findings could lay the groundwork for the advancement of gas embolotherapy.

An ex vivo study of the correlation between acoustic emission and microvascular damage.

The objective of this study was to conduct an ex vivo examination of correlation between acoustic emission and tissue damage. Intravital microscopy was employed in conjunction with ultrasound exposure in cremaster muscle of male Wistar rats. Definity microbubbles were administered intravenously through the tail vein (80microL.kg(-1).min(-1)infusion rate) with the aid of a syringe pump. For the pulse repetition frequency (PRF) study, exposures were performed at four locations of the cremaster at a PRF of 1000, 500, 100 and 10Hz (one location per PRF per rat). The 100-pulse exposures were implemented at a peak rarefactional pressure (P(r)) of 2MPa, frequency of 2.25MHz with 46 cycle pulses. For the pressure amplitude threshold study, 100-pulse exposures (46 cycle pulses) were conducted at various peak rarefactional pressures from 0.5MPa to 2MPa at a frequency of 2.25MHz and PRF of 100Hz. Photomicrographs were captured before and 2-min postexposure. On a pulse-to-pulse basis, the 10Hz acoustic emission was considerably higher and more sustained than those at other PRFs (1000, 500, and 100Hz) (p<0.05). Damage, measured as area of extravasation of red blood cells (RBCs), was also significantly higher at 10Hz PRF than at 1000, 500 and 100Hz (p<0.01). The correlation of acoustic emission to tissue damage showed a trend of increasing damage with increasing cumulative function of the relative integrated power spectrum (CRIPS; R(2)=0.75). No visible damage was present at P(r)< or =0.85MPa. Damage, however, was observed at P(r)> or =1.0MPa and it increased with increasing acoustic pressure.

An in vitro study of the correlation between bubble distribution, acoustic emission, and cell damage by contrast ultrasound.

The objective of this study was to investigate the influences of total exposure duration and pulse-to-pulse bubble distribution on contrast-mediated cell damage. Murine macrophage cells were grown as monolayers on thin polyester sheets. Contrast agent microbubbles were attached to these cells by incubation. Focused ultrasound exposures (P(r) = 2 MPa) were implemented at a frequency of 2.25 MHz with 46 cycle pulses and pulse repetition frequencies (PRF) of 1 kHz, 500 Hz, 100 Hz, and 10 Hz in a degassed water bath at 10 or 100 pulses. A 1 MHz receive transducer measured the scattered signal. The frequency spectrum was normalized to a control spectrum from linear scatterers. Photomicrographs were captured before, during, and after exposure at a frame rate of 2000 fps and a pixel resolution of 960 x 720. Results clearly show that cell death is increased, up to 60%, by increasing total exposure duration from 0 ms to 100 ms. There was an increasing difference in cell damage between a 10-pulse exposure and a 100-pulse exposure with increasing PRF. The greatest change in damage occurred at 1000 Hz PRF with a 53% increase between 10-pulse and 100-pulse exposures. For each pulse from 0 to 10, an overlay of the 2 mum bubble count with corresponding emission shows consistent behavior in its pulse-to-pulse changes, indicating a correlation between acoustic emission, bubble distribution, and cell damage.

Aligned electrospun nanofibers specify the direction of dorsal root ganglia neurite growth.

Nerve injury, a significant cause of disability, may be treated more effectively using nerve guidance channels containing longitudinally aligned fibers. Aligned, electrospun nanofibers direct the neurite growth of immortalized neural stem cells, demonstrating potential for directing regenerating neurites. However, no study of neurite guidance on these fibers has yet been performed with primary neurons. Here, we examined neurites from dorsal root ganglia explants on electrospun poly-L-lactate nanofibers of high, intermediate, and random alignment. On aligned fibers, neurites grew radially outward from the ganglia and turned to follow the fibers upon contact. Neurite guidance was robust, with neurites never leaving the fibers to grow on the surrounding cover slip. To compare the alignment of neurites to that of the nanofiber substrates, Fourier methods were used to quantify the alignment. Neurite alignment, however striking, was inferior to fiber alignment on all but the randomly aligned fibers. Neurites on highly aligned substrates were 20 and 16% longer than neurites on random and intermediate fibers, respectively. Schwann cells on fibers assumed a very narrow morphology compared to those on the surrounding coverslip. The robust neurite guidance demonstrated here is a significant step toward the use of aligned, electrospun nanofibers for nerve regeneration. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2007.

The relationship of acoustic emission and pulse-repetition frequency in the detection of gas body stability and cell death.

The effect of pulse-repetition frequency (PRF) and number of exposures on membrane damage and subsequent death of contrast agent-attached phagocytic cells was examined. Phagocytic cells of a mouse macrophage cell line were grown as monolayers on thin Mylar sheets. Optison microbubbles were attached to these cells by incubation. Focused ultrasound exposures (Pr = 2 MPa) were implemented at a frequency of 2.25 MHz with 46 cycle pulses and clinically relevant PRFs of 1 kHz, 100 Hz, 10 Hz, 1 Hz and 0.1 Hz in a degassed water bath. A 1-MHz receive transducer measured the scattered signal. The frequency spectrum was normalized to a control spectrum from linear scatterers. Photomicrographs of the cell monolayer were made before and after exposure, and a dye exclusion test (Trypan blue) was used to find the percentage of blue-stained cells indicating cell death, which was then related to acoustic emission. For 10 acoustic pulses and a high prerinse gas body concentration, there was less cell death and correspondingly lower change in the acoustic emissions at a PRF of 1 kHz than with PRFs of 100 Hz, 10 Hz, 1 Hz and 0.1 Hz (p < 0.001). The reduced effect at high PRF may be indicative of some evolution of the shelled microbubble that requires significant total exposure duration (> 10 ms, but < 100 ms).