Diane Dalecki

Enhancement of Fibrinolysis With 40-kHz Ultrasound

BACKGROUND Ultrasound at frequencies of 0.5 to 1 MHz and intensities of > or =0.5 W/cm2 accelerates enzymatic fibrinolysis in vitro and in some animal models, but unacceptable tissue heating can occur, and limited penetration would restrict application to superficial vessels. Tissue heating is less and penetration better at lower frequencies, but little information is available regarding the effect of lower-frequency ultrasound on enzymatic fibrinolysis. We therefore examined the effect of 40-kHz ultrasound on fibrinolysis, tissue penetration, and heating. METHODS AND RESULTS 125I-fibrin-radiolabeled plasma clots in thin-walled tubes were overlaid with plasma containing tissue plasminogen activator (tPA) and exposed to ultrasound. Enzymatic fibrinolysis was measured as solubilization of radiolabel. Tissue attenuation and heating were examined in samples of porcine rib cage. Fibrinolysis was increased significantly in the presence of 40-kHz ultrasound at 0.25 W/cm2, reaching 39+/-7% and 93+/-11% at 60 minutes and 120 minutes, compared with 13+/-8% and 37+/-4% in the absence of ultrasound (P<0.0001). The acceleration of fibrinolysis increased at higher intensities. Attenuation of the ultrasound field was only 1.7+/-0.5 dB/cm through the intercostal space and 3.4+/-0.9 dB/cm through rib. Temperature increments in rib were <1 C/(W/cm2). CONCLUSIONS These findings indicate that 40-kHz ultrasound significantly accelerates enzymatic fibrinolysis at intensities of > or =0.25 W/cm2 with excellent tissue penetration and minimal heating. Externally applied 40-kHz ultrasound at low intensities is a potentially useful therapeutic adjunct to enzymatic fibrinolysis with sufficient tissue penetration for both peripheral vascular and coronary applications.

Hemolysis in vivo from exposure to pulsed ultrasound

Ultrasonically induced hemolysis in vivo when a commercial ultrasound contrast agent, Albunex, was present in the blood. Murine hearts were exposed for 5 min at either 1.15 or 2.35 MHz with a pulse length of 10 microseconds and pulse repetition frequency of 100 Hz. During the exposure period, four boluses of Albunex were injected into a tail vein for a total of approximately 0.1 mL of Albunex. Following exposure, blood was collected by heart puncture and centrifuged, and the plasma was analyzed for hemoglobin concentration. With Albunex present in the blood, the threshold for hemolysis at 1.15 MHz was 3.0 +/- 0.8 MPa (mean +/- SD) peak positive pressure (approximately 1.9 MPa negative pressure, approximately 180 W cm-2 pulse average intensity). For the highest exposure levels (10 MPa peak positive pressure at the surface of the animal), the mean value for hemolysis was approximately 4% at 1.15 MHz and 0.46% at 2.35 MHz, i.e., the threshold at 2.35 MHz is > 10 MPa peak positive pressure. In contrast, hemolysis in control mice receiving saline injections at 10 MPa or sham-exposed (0 MPa) mice receiving Albunex was approximately 0.4%.

Low-intensity ultrasound increases endothelial cell nitric oxide synthase activity and nitric oxide synthesis.

Summary.  Low‐intensity ultrasound (US) increases tissue perfusion in ischemic muscle through a nitric oxide (NO)‐dependent mechanism. We have developed a model to expose endothelial cells to well‐characterized acoustic fields in vitro and investigate the physical and biological mechanisms involved. Human umbilical vein endothelial cells (HUVEC) or bovine aortic endothelial cells (BAEC) were grown in tissue culture plates suspended in a temperature‐controlled water bath and exposed to US. Exposure to 27 kHz continuous wave US at 0.25 W cm−2 for 10 min increased HUVEC media NO by 102 ± 19% (P < 0.05) and BAEC by 117 ± 23% (P < 0.01). Endothelial cell NO synthase activity increased by 27 ± 24% in HUVEC and by 32 ± 16% in BAEC (P < 0.05 for each). The cell response was rapid with a significant increase in NO synthesis by 10 s and a maximum increase after exposure for 1 min. By 30 min post‐exposure NO synthesis declined to baseline, indicating that the response was transient. Unexpectedly, pulsing at a 10% duty cycle resulted in a 46% increase in NO synthesis over the response seen with continuous wave US, resulting in an increase of 147 ± 18%. Cells responded to very low intensity US, with a significant increase at 0.075 W cm−2 (P < 0.01) and a maximum response at 0.125 W cm−2. US caused minor reversible changes in cell morphology but did not alter proliferative capacity, indicating absence of injury. We conclude that exposure of endothelial cells to low‐intensity, low‐frequency US increases NO synthase activity and NO production, which could be used to induce vasodilatation experimentally or therapeutically.

The influence of contrast agents on hemorrhage produced by lithotripter fields

Ultrasonic contrast agents greatly increase the side effects of low-amplitude lithotripter fields in mice. Using a piezoelectric lithotripter, adult mice were exposed to 200 lithotripter pulses with a peak positive pressure amplitude of 2 MPa. During the exposure period, mice were injected with approximately 0.1 mL of the ultrasonic contrast agent Albunex. For comparison, another group of mice experienced the same lithotripter exposures, but were not injected with contrast agent. Following exposures, animals were sacrificed and observed for hemorrhage in various organs and tissues. Mice exposed to the lithotripter field alone had minimal hemorrhage only in the intestine and lung. In comparison, mice injected with Albunex during exposure exhibited extensive hemorrhage in the intestine, kidney, muscle, mesentery, stomach, bladder, seminal vesicle and fat.

American Institute of Ultrasound in Medicine consensus report on potential bioeffects of diagnostic ultrasound: Executive summary

The continued examination of potential biological effects of ultrasound and their relationship to clinical practice is a key element in evaluating the safety of diagnostic ultrasound. Periodically, the American Institute of Ultrasound in Medicine (AIUM) sponsors conferences bringing experts together to examine the literature on ultrasound bioeffects and to develop conclusions and recommendations related to diagnostic ultrasound. The most recent effort included the examination of effects whose origins were thermal or nonthermal, with separate evaluations for potential effects related to fetal ultrasound. In addition, potential effects due to the introduction of ultrasound contrast agents were summarized. This information can be used to assess risks in comparison to the benefits of diagnostic ultrasound. The conclusions and recommendations are organized into 5 broad categories, with a comprehensive background and evaluation of each topic provided in the corresponding articles in this issue. The following summary is not meant as a substitute for the detailed examination of issues presented in each of the articles but rather as a means to facilitate further study of this consensus report and implementation of its recommendations. The conclusions and recommendations are the result of several rounds of deliberations at the consensus conference, subsequent review by the Bioeffects Committee of the AIUM, and approval by the AIUM Board of Governors.

THRESHOLDS FOR ULTRASONICALLY INDUCED LUNG HEMORRHAGE IN NEONATAL SWINE

The threshold for generation of lung hemorrhage in adult mice by pulsed ultrasound has been shown to be approximately 1 MPa at the surface of the lung (10-microseconds pulse and a carrier frequency of 2 MHz). This investigation used neonatal swine to determine if the findings for mice can be generalized to other species. After exploratory observations, the inverse sampling method was used in a primary study (22 animals, 88 exposure sites) to determine the threshold for lung hemorrhage in neonatal swine. The primary study was followed by a separate confirmation study (13 animals, 48 exposure sites), testing the conclusions of the first study and comparing damage at subthreshold levels with sham-exposed animals. A separate investigation explored the histological nature of tissue damage at suprathreshold levels. A 2.3-MHz focused transducer (10 microseconds at 100-Hz pulse-repetition frequency) was incremented vertically for a distance of 2 cm over the chest of the subject for a total exposure period of 16 min. Animals were euthanized and lungs were scored by visual inspection for numbers and areas of gross hemorrhages. The threshold level for hemorrhage was approximately 1.5 MPa peak positive pressure in water at the surface of the animal or, at the surface of the lung, 1.1 MPa peak positive pressure, 1 MPa fundamental pressure, 0.9 MPa maximum negative pressure, 25 W cm-2 pulse average intensity or a mechanical index of 0.6. These values are essentially the same as those reported for adult mice.

Tactile perception of ultrasound.

In this investigation, acoustic radiation force was used as a stimulus to determine the threshold for tactile perception in the human finger and upper forearm as a function of frequency and pulse duration. Initially, a small (1.8-cm2) acoustically reflecting disk was affixed to the anatomical exposure site to maximize the delivered radiation force. Exposures were performed using a 2.2-MHz unfocused source modulated to produce square waves at 50, 100, 200, 500, and 1000 Hz. For the finger, maximum tactile sensitivity occurred at 200 Hz with a threshold radiation force of approximately 0.4 mN. For single pulses of 1 to 100 ms at 2.2 MHz, the threshold forces were an order of magnitude greater than for continuous exposure modulated at 200 Hz. Thresholds for pulse durations of 0.1 ms were somewhat greater than for pulses longer than 1 ms. Subsequently, thresholds of tactile perception were determined for direct exposure of the upper forearm (avoiding bone) to single pulses of 2.2-MHz ultrasound. Comparison of perception thresholds with and without a reflecting material over the tissue were consistent with the hypothesis that the tactile sensation experienced when tissue is exposed to ultrasound is its response to the radiation force associated with the transfer of momentum from the sound field to the tissue medium.

Age dependence of ultrasonically induced lung hemorrhage in mice.

Thresholds for ultrasonically induced lung hemorrhage were determined in neonatal mice (24-36 h old), juvenile mice (14 d old) and adult mice (8-10 weeks old) to assess whether or not the threshold for lung hemorrhage is dependent upon age. Ultrasonic exposures were at 1.15 MHz with a pulse length of 10 microseconds, pulse repetition frequency of 100 Hz and a total exposure duration of 3 min. The threshold for lung hemorrhage occurred at a peak positive acoustic pressure of approximately 1 MPa for mice in all three age groups. Although the thresholds were similar for neonatal, juvenile and adult mice, the sizes of the suprathreshold hemorrhages were significantly larger in adult mice than in neonatal or juvenile mice.

Ultrasonically induced lung hemorrhage in young swine

Ten-day old swine were used in the final step of a study of the age dependence of the threshold for lung hemorrhage resulting from exposure to diagnostically relevant levels of pulsed ultrasound. A 2.3-MHz focused transducer (pulse length of 10 microseconds, 100-Hz pulse repetition frequency) was incremented vertically at several sites for a distance of 2 or 2.5 cm over the chest of the subject for a total exposure period of 16 or 20 min. The procedure was repeated at a total of four sites per animal. Animals were euthanized and lungs were scored by visual inspection for numbers and areas of gross hemorrhages. The threshold level for hemorrhage was approximately 1.3-MPa peak positive pressure in water and the surface of the animal or, at the surface of the lung, 0.8-MPa peak positive pressure, 0.8-MPa fundamental pressure, 0.7-MPa maximum negative pressure and 20 Wcm-2 pulse average intensity. These values are essentially the same as those reported previously for neonatal swine, and neonatal, juvenile and adult mice.

Effects of pulsed ultrasound on the frog heart: III. The radiation force mechanism

Earlier studies have shown that a single, millisecond duration pulse of ultrasound delivered to the frog heart in vivo during systole can produce a reduction in the developed aortic pressure, while a pulse delivered during diastole can produce a premature ventricular contraction. The threshold for these effects is 5-10 MPa with a 5-ms pulse. Since cardiac tissues respond to mechanical stimulation, the objective of this study was to investigate acoustic radiation force as a possible mechanism for the observed effects of ultrasound on the frog heart. In two experiments, the radiation force exerted on the heart was varied by varying the ultrasonic frequency and the acoustic beam width. Results of these studies indicated that the rate of occurrence of the reduced aortic pressure effect was directly correlated with the magnitude of the radiation force exerted on the heart. A third experiment tested the radiation force mechanism directly by placing an acoustic reflector on the frog heart. The acoustic reflector maximized the radiation force delivered to the heart, but eliminated direct interaction of the ultrasound with the heart and experimentally eliminated heating and cavitation as mechanisms of action. The reduced aortic pressure effect was observed with the reflector on the heart, indicating that radiation force is capable of producing this effect. No premature ventricular contractions were observed with the acoustic reflector over the heart, suggesting that another property of the exposure may be responsible for this bioeffect.