Current status of research on biophysical effects of ultrasound

Ultrasound in Med. & Biol., Vol. 20, No. 3, pp. 205-218, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0301-5629/94 $6.00 + .00

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CURRENT STATUS OF RESEARCH ON BIOPHYSICAL EFFECTS OF ULTRASOUND S. B. BARNETT, t G. R. TER HAAR, * M. C. ZISKIN,* W. L. NYBORG, § K. MAEDA II and J. BANG¶ +Ultrasonics Laboratory, CSIRO, Division of Radiophysics, 126 Greville Street, Chatswood 2067, Australia; •Joint Department of Physics, Institute of Cancer Research and Royal Marsden Hospital, Sutton, Surrey SM2 5PT, UK; *Centre for Biomedical Physics, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140, USA; ~Department of Physics, University of Vermont, Burlington, VT 05405-0125, USA; IIDepartment of Obstetrics and Gynecology, Seirei Hammarnatsu Hospital, Sumiyoshi 2-12-12, Hammamatsu 430, Japan; IDepartment of Diagnostic Ultrasound, Rigshospitalet, Copenhagen, Denmark (Received and in final form 10 December 1993)

Abstract--This overview of bioeffects of ultrasound presents some key aspects of selected papers dealing with biophysical end-points. Its purpose is to establish a basis for exposure and dosimetric standards for medical ultrasonic equipment. It is intended to provide essential background resource material for the medical/scientific community, and more specifically for scientific working groups. This document was prepared by members of the Safety Committee of the World Federation for Ultrasound in Medicine and Biology. It was produced as a resource document in response to a request for information by Working Group 12 (Ultrasound exposure parameters) of the International Electrotechnical Commission Technical Committee 87, Ultrasonics. IEC TC 87, WG12 is the working group responsible for generating international standards for the classification of equipment by its acoustic fields based on safety thresholds. Our paper is intended to update and supplement information on the thermal mechanism provided in the publication, "WFUMB Symposium on Safety and Standardisation in Medical Ultrasound: Issues and Recommendations Regarding Thermal Mechanisms for Biological Effects of Ultrasound" (WFUMB 1992). It also provides an overview of trends in research into nonthermal mechanisms as a prefiminary to the next WFUMB Symposium on Safety of Medical Ultrasound when this subject will be examined in detail by a select group of international experts. The WFUMB-sponsored workshop will take place in Utsunomiya, Japan during l l - 1 5 t h July, 1994. The purpose of the meeting is to evaluate the scientific literature and to formulate internationally accepted recommendations on the safe use of diagnostic ultrasound that may be endorsed as official policy of the WFUMB. It should be noted that the current publication is not intended for review or endorsement as an official WFUMB document. It is produced as a scientific paper by individuals who are members of the WFUMB Safety Committee, and it therefore represents the opinions of the authors. Nevertheless, during the preparation of this document, contributions were received from members of the International Electrotechnical Commission Technical Committee 87 as well as many other individual experts, and the authors sincerely acknowledge their support.

Key Words: Ultrasound, Ultrasonic tissue effects, Biological effects, Mechanisms, Temperature increase, Cavitation, Ultrasound safety. INTRODUCTION

sound has encouraged its widespread application in medicine. However, the improvements in diagnostic sensitivity have been accompanied by substantial increases in the acoustic output levels of ultrasonographic equipment, especially in pulsed Doppler applications. It is important to realize that the past safety record should not be mistaken for a guarantee that harm can never occur in the future. It is the duty of health care providers, as well as health care regulators, to remain vigilant to reduce the possibility of any harm from exposures from current or new applications.

Ultrasonographic imaging has been used clinically as an effective diagnostic tool over the past 30 years. In spite of literally millions of examinations, there is no verified documented epidemiologic evidence of adverse effects in patients caused by exposure to ultrasound (Ziskin and Petitti 1988). The perceived safety of diagnostic ultraAddress correspondence to: S. B. Barnett. The authors are members of the Safety Committee of the World Federation for Ultrasound in Medicine and Biology. 205

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Laboratory studies have shown that ultrasound is definitely capable of producing serious biological damage if the intensity is sufficiently high. The damage can result from thermal or nonthermal mechanisms. Both mechanisms are important, and either may predominate depending on the exposure conditions. Knowledge of the mechanisms involved is important to extrapolate research findings in laboratory conditions to predictions of effects from human exposures. This report provides a survey of the current understanding of biological effects resulting from exposure to ultrasound. Both thermal and nonthermal mechanisms are covered. Special consideration is given to those aspects that relate most directly to human safety. It is intended that this information will be of benefit to manufacturers of ultrasound equipment as well as those who use ultrasound in patient examinations, and to those who set standards of practice and who regulate the use of ultrasound in medicine. TEMPERATURE This section presents the current level of understanding of the biological consequences of temperature elevation in both adult and fetal tissues. The bulk of the information relates to the effects of hyperthermia in the absence of ultrasound. The principal aim is to provide sufficient information to allow the development of models to predict biological effects due to ultrasound absorption. Temperature increases within the human body may be due either to fever or to hyperthermia. There is a difference between these two phenomena in that fever results in an elevation of the body's "set point" for thermo-regulation whereas hyperthermia does not, even though temperatures well above that level may be achieved. Fever is thought to be an adaptive reaction to viral or bacterial infection that is effective because many infective agents do not tolerate high temperatures. It is accompanied by the production of an endogenous pyrogen which is released by leucocytes and shifts the body's "thermostat" to a higher level. Hyperthermia does not lead to pyrogen production but, when the temperature exceeds a certain level, it may induce shock proteins. Hyperthermia may result from exposure to hot water, hot air or sunshine. Local heating is used in cancer therapy to produce hyperthermia and thus to suppress tumour growth. In contrast to environmentally induced hyperthermia, the absorption of ultrasound, microwave or radio-frequency energy leads to a comparatively rapid temperature rise which is localised to the area of the beam. Absorption of ultrasound, leading to rapid heating on the time-scale of seconds would bypass any possible thermo-protec-

Volume20, Number 3, 1994 Table 1. Mean rectal temperatures of some mammals. Fetal temperature generally exceeds matemal core temperature by approximately 0.5°C. Species

Temp. °C

Human Mouse, monkey Rat, cattle Dog, pig Rabbit, guinea pig, sheep Goat

37.0 38.0 38.5 39.0 39.5 40.0

tive mechanism afforded by heat shock proteins, which require many minutes of exposure to elevated temperatures (15 min above 42°C) for induction (Walsh et al. 1987).

Temperature variation in mammals The human body normally maintains a nearly constant core temperature (in the brain, heart, kidney, fetus) that is achieved by complex metabolic and neural mechanisms which balance heat loss and internally generated heat. The variability of this "constant" body core temperature is limited to a few degrees in either direction. Adults in excellent health have been observed to exhibit diurnal core temperature variations ranging from 0.35 ° to 1.0°C from the mean (Hardy 1982). Skin temperatures can vary from 31.4°C at the calf to 34.5°C at the abdomen. Due to its high metabolic activity, the liver has a normal temperature of 39°C. These values refer to the nonactive, resting state (Hales 1984). Vigorous exercise can raise the core temperature to above 40°C, and a core temperature of 41°C has been reported in a marathon runner (Maron et al. 1977). Although the pathology has not been studied, it is well known that these athletes require substantial rest and recovery periods between races. Body temperature also varies considerably amongst other homeothermal species. Table 1 shows a range of 37 ° to 40°C for normal body temperatures in humans and goats. When determining safety on a thermal basis from animal studies, it is important to realise that a temperature increase to 41°C in the guinea pig represents a rise of only 1.5°C, whereas this temperature in the human is equivalent to a rise of 4°C above normal body temperature. Physiological reactions to elevated temperatures In humans the reaction to increased core temperature up to 40.5°C is reversible. The resulting physiological changes vary from organ to organ, and are shown in Table 2. Clinical reactions to sustained hyperthermia at this level include circulatory collapse, muscular heat cramps caused by salt deficiency, and exhaustion. Tis-

Biophysical effects of ultrasound • S. B. BARNETT et al.

Table 2. Reversible human physiological reactions associated with elevated core temperatures to 40.5°C. Physiological parameter

Response

Metabolic activity Skin Heart Bowels Endocrine system Brain

Increase by 7% for each 0.5°C Vasodilatation, increased sweat production Increased heart rate and cardiac output Vasoconstriction Increased cortisol production Increased susceptibility to epileptic seizures

sue and organ damage becomes increasingly probable. A sustained temperature above 41.5°C is barely compatible with life. Disseminated necroses of renal tubular epithelia and heart muscle fibres, and fatty degeneration of liver cells have been described. Similar changes, and oedema, are found in the brain. In addition, these organs show disseminated bleeding, increased blood viscosity and reduced coagulation capability. Clinical consequences include renal failure and loss of consciousness. A temperature above 43°C is an ominous warning of death by heat stroke. Table 3 illustrates the range of temperatures, measured orally, associated with fever.

Biological effects of elevated temperature Biological structures are naturally exposed to a wide range of "physiological" temperatures, beyond which the life of an organism cannot be sustained. For example, in vitro mammalian cells will die if frozen at 0°C, and have cell growth kinetics which appear maximal at 33-39°C. Extended exposure to temperatures above 42°C may reduce cell survival rates. The magnitude of the effect varies with the exposure duration. Cells are most sensitive to heat during the process of mitosis. If mitosis in neurones is arrested by a transient temperature increase during embryonic development, the resulting neural deficit may not be restored, although the fetus may continue to develop and appear morphologically normal (Edwards et al. 1974). At temperatures above 45°C proteins become denatured, i.e., they lose their tertiary structure, enzymes are no longer able to support vital chemical reactions (Miller and Ziskin 1989) and cell death occurs. It is well known that large temperature elevations (exceeding 6°C) result in cell death. However, smaller elevations are capable of causing significant biological effects. These can be widespread and include such reactions as: increased metabolic activity and cell cycling rates, increased heart rate and altered blood perfusion rates, increased leakage of proteins through capillary membranes, edema formation, and the production of heat shock proteins.

207

Experimental studies have shown that increased temperature has led to developmental defects in chickens, mice, rats, hamsters, guinea pigs, rabbits, sheep, pigs, and nonhuman primates. The methods of heating have typically involved placing a pregnant mother in a hot air chamber (incubator) or in heated water. Experiments are usually performed for a predetermined heat stress period (10 min to 30 h), rather than at a specific fetal temperature. As a result, actual fetal temperatures are not known for most studies. Furthermore, a substantial amount of time is required to overcome the maternal homeothermic response so that the duration of exposure to the peak temperature in the fetus is also unknown.

Embryos and fetuses. Fetal temperature generally exceeds the maternal core temperature by 0.5-1.0°C. The embryos and fetuses of all animal species, including humans, are susceptible to increases in temperature. The effects produced depend on the stage of embryonic and fetal development, and the level and duration of the temperature elevation. Embryonic or fetal death commonly follows exposure to heat at preimplantation stages, during organogenesis, and at fetal stages of development. Lethal effects can be caused at developmental stages that are not usually associated with teratogenesis, as well as at stages that are sensitive to teratogenic change by heat. The cause of death appears to be a direct lethal effect of heat on embryonic cells. When the temperature elevation is due to maternal hyperthermia, placental infarction can occur. Uterine activity is increased during maternal hyperthermia and this can result in abortion. Developmental effects caused by temperature elevation in animals include anencephaly/exencephaly, encephalocele, microphthalmia, micrencephaly, midface hypoplasia, brainstem palsies, facial clefting, talipes, arthrogryposis, micromelia, exomphalos, and disorders of muscle tone and locomotion. Many of these defects have also been found in children following in utero febrile episodes. The type of defect produced depends largely on the animal species, the developmental stage during exposure, and the " d o s e " of heat delivered to the embryo. The developing central ner-

Table 3. Health classification of oral temperature. Condition

Temp. °C

Normal Febricular Mild fever Average fever High fever Severe fever

37.0 38.0 38.5 39.5 40.5 42.0

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vous system is particularly sensitive in all species. Nondeforming retardation of brain growth with reduced learning performance are amongst the most common abnormalities found in heat exposed guinea pig offspring (Edwards 1988), and these defects can be caused during both early and later fetal growth. In general, embryos are more susceptible to damage than the more developmentally advanced fetuses. Studies with systemic heating have shown neural tissue damage (Edwards 1969, 1986) and micrencephaly (brain growth retardation) in guinea pigs following maternal temperature elevations of 1.5° to 2.5°C for 1 h at 21 days gestational age (dga). Furthermore, each I°C increase above the threshold produced an additional 8% increase in the incidence of micrencephaly. Increased abortion rates have been reported in monkeys exposed to temperature elevations of 2.6 ° to 3.9°C for 72 min, between 21 and 45 dga (Hendrickx et al. 1979). Monkeys have also shown fetal growth retardation following a 4.5°C increase in maternal temperature, achieved by one-hourly incubation on five days between the 25th and 50th dga (Poswillow et al. 1974). For the most sensitive period in fetal development in guinea pigs (20-23 dga), the threshold for teratological effects was 1.5°C elevation at the fetus (Edwards 1969). This is described in Chapter 1 of the report of the 1991 WFUMB Symposium on Safety and Standardisation of Medical Ultrasound (WFUMB 1992). In studying the effect of the duration of hyperthermia, Germain et al. (1985) showed that for rat embryos, a 4.5°C "heat spike" (43°C for 7.5 min) was teratogenic (producing microphthalmia), while progressively longer exposures required smaller temperature elevations to produce the same abnormality. At the lower temperature elevation of 2.5°C, the time threshold for maternal whole-body exposure was 60 min. Proliferating neuro-epithelial cells are especially sensitive to damage by heat, and death of these cells is probably the basis for defects of neural tube closure, microphthalmia and micrencephaly. Microvascular disruption caused by damage to the endothelium may be the basis for a number of other defects. Embryo culture techniques (Cockroft and New 1978) have allowed the study of the embryo isolated from the maternal environment. Early studies showed that temperature elevations of 2°C for 48 h during the 9th to 10th gestational day resulted in disruption of head and brain development (Cockroft and New 1978).

Local hyperthermia. Local hyperthermia can induce lesions in the surviving patient. In the skin, a temperature above 47°C induces erythema and pain, above 55°C (40 s) blistering, and above 60°C (60 s) necrosis. The threshold level for irreversible damage

Volume20, Number3, 1994 of fatty tissue, muscle, cartilage and gut is 43°C. A study on ultrasound-induced heating of the femur bone marrow in adult guinea pigs (Barnett et al. 1991) reported irreversible changes involving hypersegmented nuclei in neutrophils following localised exposure to 43°C for 3 min. Similar nuclear abnormalities were observed after longer exposures to the whole body when guinea pigs were heated in air incubators for 60 min (Edwards and Penny 1985). For the guinea pig species this threshold represents an increase of 3.5°C above normal body temperature. While changes at the cellular level have been observed for temperature variations of as little as 0.5°C, most adults have experienced increases in local peripheral temperature of up to 4°C without any lasting harmful effect. The body is able to tolerate local transient temperature-induced changes in reaction rates, and to function normally. However, this is not the case for the developing fetus where temperature elevation is a well-known teratogenic agent (Edwards 1986, 1993). The effects seen in offspring following hyperthermic embryopathy (whether viral-induced or other) are similar for children and experimental animals (Jones 1988). In therapeutic hyperthermia, the tissue temperature is intentionally increased locally to exceed 42°C in order to heat sensitive cancer cells while sparing neighbouring normal cells. However, of greatest concern for the safety of ultrasound in clinical diagnosis is the ability of smaller temperature elevations to produce fetal abnormalities (Lele 1975). Interrogation of biological tissues by an ultrasound beam, during a diagnostic examination, delivers peak energy levels to a narrow, localised region. The extent of heating resulting from the absorption of this energy depends on both the acoustic and biological properties. Materials such as bone, teeth and optic lenses have high acoustic absorption coefficients, and are heated to a greater extent than soft tissue. The absorption coefficient of bone is at least 30 times greater than for soft tissue (NCRP 1992). Tissues lying close to, or in contact with bone will be significantly heated by conduction. The most sensitive target for ultrasound-induced biological effects is the central nervous system of the developing embryo and fetus. During the embryonic stage of development (up to 56 days from fertilization in the human) the soft tissue mass has low absorption characteristics and is unlikely to be at risk from a thermal mechanism, although it is sensitive to many physical agents and nonthermal mechanisms. As bone develops so does the extent of ultrasound-induced heating and the potentia/ risk of biological effects in the actively proliferating neural tissue of the central nervous system (CNS). As a result of the 1991 Symposium on the safety

Biophysical effects of ultrasound • S. B. BARNETT et al.

of medical ultrasound (WFUMB 1992), the WFUMB has adopted, as official policy, a statement reflecting international consensus on thermal effects in pulsed Doppler clinical applications, which is as follows: "It has been demonstrated in experiments with unperfused tissue that some Doppler diagnostic equipment has the potential to produce biologically significant temperature rises, specifically at bone/soft tissue interfaces. The effects of elevated temperatures may be minimized by keeping the time for which the beam passes through any one point in tissue as short as possible. Where output power can be controlled, the lowest available power level consistent with obtaining the desired diagnostic information should be used. Although the data on humans are sparse, it is clear from animal studies that exposures resulting in temperatures less than 38.5°C can be used without reservation on thermal grounds. This includes obstetric applications."

Threshold temperature elevations A quantitative relationship developed by Sapareto and Dewey (1984) between the temperature elevation and exposure duration needed to cause cell death was shown to apply also to hyperthermia-induced fetal abnormalities (Miller and Ziskin 1989). This relationship is given by: t43 = t ' R (43-T)

where T is the temperature producing an abnormality, t is the duration of the exposure producing that abnormality, and t43 is the duration that would have been necessary to produce the abnormality had the temperature been 43°C. The value of R is 0.25 for temperatures below 43°C and 0.5 for temperatures above 43°C. No thermally induced fetal abnormalities have been observed for temperature-time combinations where t43 < 1 (Miller and Ziskin 1989). This relationship shows that the severity of a thermal injury is directly proportional to the logarithm of the exposure duration. There is a lower limit to the temperature-time relationship as there is an absence of measured significant, or lethal, effects below 41°C. The threshold elevation of temperature for rodent fetal malformation and retardation of head and brain growth exceeds 1.5°C (Edwards 1986, 1993) when pregnant mothers were heated for 60 min. However, the threshold for duration has not been determined for small increases in temperature. Exposures for 60 min were commonly used to overcome the homeothermic response of the mother, and are not directly relevant for extrapolation to the effects of immediate ultrasoundinduced heating. Different defects result from different " d o s e s " of heat. Retardation of the growth of the brain and head, microphthalmia, neural tube defects and maxillary hypoplasia appear to have the lowest thresholds in experimental animals. Higher temperature elevations require much shorter exposures to cause defects than lower elevations. Based on a review of the

209

literature on hyperthermia (WFUMB 1992), it is probable that a temperature elevation of up to 1.5°C will not cause defects of development, even when exposures are prolonged. The threshold elevation of temperature associated with embryonic death is not as clearly defined. Most reports indicate that embryonic death is associated with elevations exceeding 1.5°C over prolonged periods (WFUMB 1992).

Potentiating effect of ultrasound exposure Most teratogenic studies consider the effects of exposure to heat alone. In studies on rat embryos in culture, adverse effects of heating and ultrasound have been observed in the absence of maternal influence. A common finding associated with hyperthermia is that of reduced size of the prosencephalon with a threshold " d o s e " of 43°C (elevation of 4.5°C in rats) for 7.5 min (Walsh et al. 1985). Using the same experimental protocol, Barnett et al. (1990) found no effect on brain development at 40°C for 30 min. However, when pulsed ultrasound (1.2 W/cm 2 lserg) was applied for 15 min at 40°C (1.5°C above normal) reduced brainbody ratio and delayed embryonic development was observed. Production of heat shock proteins and oedema of forebrain in rat embryos was also reported after 15 min exposure to the same ultrasound conditions plus 1.5°C temperature increase (Angles et al. 1990), whereas the threshold for temperature increase in the absence of ultrasound was 3.5°C increase for 15 min (Walsh et al. 1987).

Outstanding problems There is not yet a clear consensus on whether absolute temperature or temperature elevation is the most biologically relevant parameter. A suggested clinical approach is to avoid temperatures exceeding 38.5°C. The possible influence of potentiating factors should be considered and special care should be taken when scanning febrile pregnant mothers. Most of the literature deals with whole-body heating. The biological consequence of heating small local areas for short time-intervals has not been adequately addressed experimentally. ACOUSTIC CAVITATION Cavitation has been defined in the literature as: The "formation and/or activity of gas or vapour filled cavities (bubbles) in a medium exposed to an ultrasonic field" (ter Haar 1986).

Generally accepted terms for specific aspects of bubble activity exist, and their descriptions are: Stable cavitation describes the continuous oscillation of bubbles in response to alternating positive and negative pressures in an acoustic field. The bubble ra-

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dius varies about an equilibrium value, and the pulsating cavity exists for a considerable number of cycles. Stable cavitation in liquid media leads to acoustic streaming and can result in high shear stresses. Inertial (collapse) cavitation (formerly known as transient cavitation) describes the behaviour of bubbles that oscillate about their equilibrium size, grow until the outward excursion of the surface exceeds a limiting value (approximately 2 × the initial radius, Flynn 1982), and collapse violently. This can occur during the period of a single cycle (Fowlkes and Crum 1988) or a few cycles, depending on the applied acoustic pressure and the properties of the propagating medium. High temperatures and pressures are associated with the collapse phase. When a sufficiently high temperature is reached (>1500°K), the released energy may cause the emission of light and the formation of reactive chemical species. The region occupied by an imploding bubble is of the order of tens of microns and the time-scales of the events are microseconds. Although enormous amounts of energy are deposited (approx. 100 MeV, Apfel 1986) the effects are instantaneous and highly localised. Thus, the effects of such events are likely to be most important in the early stages of embryonic development and organ differentiation where damage to single cells or small groups of cells may be devastating. For asymmetrical bubble motion, close to a rigid boundary, the effects may be more pronounced due to the formation of a liquid jet passing through the bubble and striking the boundary at high velocity.

Biological effects of acoustic cavitation Theoretical predictions indicate that stable cavitation can lead to membrane rupture (Lewin and Bjorno 1981). If cell membrane rupture followed such insonations, its clinical significance will depend on the cell type, and on the stage of development of the tissue target. A summary of cavitation-related biological effects is given in Table 4. Ultrasound-induced gas bubble production has been demonstrated in agar gels (Daniels et al. 1987). Bubble growth has also been reported in vivo following exposure to continuouswave ultrasound at a frequency of 0.75 MHz (ter Haar and Daniels 1981). Such therapeutic exposures can generate substantial temperature increases in biological tissue, and lead to bubble formation, so the mechanism of bubble production may not yet be clearly established. It is, therefore, uncertain how this information can be extrapolated and applied to diagnostic, clinical ultrasound practice. With microsecond pulses, cavitation effects are most likely to be associated with bubble collapse, such as mechanical erosion, bubble liquid jetting, or genera-

Volume20, Number3, 1994 tion of free radicals and sonochemistry (Suslick 1988). It is less likely that such short pulses would result in stable cavitation, where the bubble needs to behave like a driven nonlinear oscillator. However, some pulsed Doppler devices may have used sufficiently high pressure amplitude, together with pulse lengths and pulse repetition frequencies that can support high amplitude stable oscillations in those bubbles with radii that do not undergo transient collapse. The mechanisms associated with such bubble activity include microstreaming and subsequent shear stresses, or bubble-related radiation forces resulting in particle aggregation. The biological effects resulting from ultrasonic exposure depend on both the acoustic and tissue properties. This review paper aims to provide a current assessment of biological effects of ultrasound which are (a) apparently due to cavitation, and (b) relevant to the assessment of potential adverse effects on human health due to ultrasonic exposure during diagnostic examinations.

Cells in suspension. It has been stated that most biological effects observed in vitro in cell suspensions are due to nonthermal mechanical processes (NCRP 1983). When cavitational activity occurs at sufficiently violent levels the primary outcome is cell death (Miller 1985), whether due directly to interaction with oscillating bubbles or indirectly as a consequence of stresses from acoustic microstreaming. Reports of genetic effects are rare, although mutagenic effects have been observed under conditions which favour violent cavitation (Doida et al. 1990, 1992; Kaufman 1985; Miller et al. 1991a). Less aggressive cavitation activity can give rise to temporary changes in ion concentrations within cells, possibly through a simple stirring mechanism (Dyson 1985). Evidence of enhanced uptake, and hence cytotoxicity, of anticancer drugs in vitro (Saad and Hahn 1989) implicate ultrasound-induced changes in cell membrane permeability. Similar effects have also been reported in vivo (Harrison et al. 1991) following exposure to pulsed ultrasound for 1 h at a peak rarefactive pressure (Pr) amplitude 0.28 MPa. The possible existence of intracellular cavitation has been implicated (Inoue et al. 1989). It is well known that an effect of collapse cavitation is the dissociation of molecules into highly reactive free radical byproducts (Suslick 1988); hence opportunities are created for biochemical change, such as single-strand chromosome breaks (Miller et al. 1991b) within ceils (Table 4). These results suggest that cell death, metabolic concentration changes, and mutagenicity may result from ultrasound-induced cavitation. Insects and plants. Cell lysis in Elodea leaves (Miller 1987) and delayed death of Drosophila melanogaster (fruit fly) larvae (Carstensen 1987) are two

tO

3.50

Tissue regeneration

CW CW

1.0

1.0 1.0-8.0

0.5 - 1.6

Free radicals detected in aqueous sols

Hind limb paralysis in mouse neonate Necrosis in cat brains

Cavitation in blood CW

6.5/~s

CW

1.61

Chromosome singlestrand breaks

CW CW

10 #s

1.2 1.0 1.0

1 #s

2.50

1 /as pulse

2 ms

Physiotherapy 2 ms pulses CW 100 #s

CW

Mutagenicity

Drosophila larvae killed Lung damage in mice

shock wave

0.75 0.75

Bubble growth in gels

Kidney damage

0.75

Frequency MHz

Bubble growth in guinea pig hind limb

Effect

Pulse duration or continuous wave

lsvrA = 16 W/cm 2

lsvra -> 500 W/cm 2 in situ

IsvrA = 2.5 W/cm 2 lsppA = 90 W/cm 2 Pr= 2MPa lsvrA = 289 W/cm 2

ISPTA = 8 W/cm 2

IsvrA = 0.1 W/cm 2 Temp. max. effective Int = 0.5 W/cm 2 Pc = 100 MPa Pr = 10 MPa ISVrA = 3 mW/cm z ISppA = 50 W/cm z Pc = 0.7 MPa ISPTA = 1 mW/cm 2 lSPTA = 35 W/cm: lsvrA = 35 W/era 2

Temp. avg. effective Int = 110 mW/cm 2 Temp. max. effective Int. = 240 mW/cm 2 Pr = 65 kPa Pr = 1 MPa

Exposure parameters

0.5 s

500 ms

10 min

10 or 30 min

10-180 s 120 s

3.0 min

2.5 min

1500 shocks in 15 rain

5 min 3 per week

1-5 min 5 min

7 - 7 0 min

Exposure time

2.6%

0.1-0.01%

0.06%

20%

50%

50%

Duty factor

T a b l e 4. S u m m a r y o f r e l e v a n t c a v i t a t i o n - r e l a t e d bioeffects.

Gross et al. 1985

Carstensen et al. 1974

FrizzeU et al. 1983

Christman et al. 1987

Kaufman 1985; Doida et al. 1990, 1992 Miller et al. 1991b

Child et al. 1990

Carstensen 1987

Delius et al. 1988

Dyson et al. 1970

Daniels et al. 1987

ter Haar et al. 1986

Reference

Bubble growth threshold for CW; pulsed exposure is below threshold Physical therapy; beneficial effect due to stable cavitation In vivo, in dogs; robust effect Indicates in vivo conditions which diagnostic ultrasound intensities may cause cavitation effects Rotating test tube; effect due to enhanced cavitation Cavitation-induced toxic sonochemicals responsible for effect Detected in aqueous solutions in vitro, not observed in vivo Threshold for paralysis in mouse In vivo focal lesions; robust effect;/2t intensity/time constant for threshold Bubbles not detected in canine flowing blood, although produced in vitro in water

Bubbles observed ultrasonically after 30 min

Remarks

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Ultrasound in Medicine and Biology

important cavitation-related biological effects that have been thoroughly studied. A common factor in the production of damage is that stresses are induced when the medium constrains the growth of bubbles within a biological structure such as a cell wall or a trachea. The relevance of these findings is that they show that, in a living system, if a collection of stabilised gaseous pockets is sufficiently extensive, death of cells or even the whole organism is possible at low ultrasonic pressures. These effects accompany exposures to microsecond length pulses with similar output parameters to those found in the great majority of diagnostic systems sold today. It may not be possible to directly extrapolate these results to mammalian systems, where the characteristics of cavitation nuclei are unknown. Mammals. The search for cavitation effects in mammalian systems has been largely hampered by the fact that, for similar ultrasonic exposures, thermal effects often mask cavitation effects. This situation has changed with the advent of pressure pulse lithotropsy. In fact, cavitation is a suspected mechanism in canine lung damage observed in experimental studies on gallstone destruction (Delius et al. 1987). Nonetheless, with ultrasound exposures, cavitation-related events have been implicated in a number of effects on mammals (Table 1) including: (a) lesions in lung tissue of adult mice (Child et al. 1990, O'Brien and Zachary 1993), (b) focal lesions of the brain (Carstensen et al. 1974), (c) irreversible hind limb paralysis of neonatal mice (Borrelli et al. 1968; Frizzell et al. 1983), and (d) therapeutic effects involving tissue, bone or blood vessel repair stimulation (Dyson 1985). The examples of destructive effects clearly indicate the vulnerability of mammalian tissues to cavitation effects once the threshold has been reached. It is not known whether the type of cavitation giving rise to repair stimulation in injured tissue could also give rise to adverse effects in, say, developing fetal tissue. The first finding of haemorrhage of lung tissue in mice was reported at a threshold of 1 MPa for pulsed ultrasound exposures (Child et al. 1990). A subsequent investigation has shown the effect to be largely dependent on the pressure amplitude threshold (Hartman et al. 1990). A n in vivo study using a resonant bubble detector reported that stable cavitation could not be detected in circulating blood in the canine abdominal aorta (Gross et al. 1985). Cavitation thresholds

Although no generally acceptable definition exists, it would be most useful to have one that indicates the minimum acoustic pressure at which "cavitation"

Volume20, Number3, 1994 first occurs. But, of necessity, the measured threshold is determined by the sensitivity of the detection technique. Thus, published in vivo values are thresholds for detectable effects of bubble collapse, such as the appearance of histological damage. In vitro methods often rely on detection of subharmonic emissions resuiting from bubble activity. No allowance is made for either the possibility of bubble growth to achieve resonant size from a micronucleus, or the preexistence of gas-filled cavities approximating resonant size. Exposure thresholds are affected by the acoustic properties (acoustic working frequency) and pulsing conditions. At least for in vitro exposures, the important parameters appear to be peak negative (rarefaction) acoustic pressure (Pr) and the acoustic pulse waveform. Pulse duration has a weak effect up to 5 cycles, and pulse repetition frequency is of lesser importance. Properties of the propagating medium such as gas content, viscosity, surface tension and temperature affect the content and distribution of nuclei essential to initiate bubble formation and activity. There is currently inadequate evidence available to define "cavitation thresholds" in vivo. Acoustic output measurements reported by Duck (1989) indicate that the peak rarefactional pressure amplitudes (Pr) for Doppler systems range from 0.2 to 6.3 MPa. Such high rarefactional pressure amplitudes are sufficient to lead to adverse biological effects. A recent study (Child et al. 1990) reported a threshold for haemorrhage in mouse lungs of 1 MPa in 10 #s pulses at 1 MHz frequency. It has since been established that the lung has an efficient mechanism for dissipating heat (Hartman et al. 1992). This provides the first experimental data of a nonthermal effect in vivo following exposure to diagnostic pulsed ultrasound. Using electron microscopy, it has been shown that the damage produced in inflated mouse lungs was limited to the period of insonation (Penny et al. 1993). The pulse repetition frequency and temporally averaged intensity that produced lung lesions were below those used in commercial Doppler devices. The same effect was reported in lungs of monkeys exposed to 3.7 MPa using a commercial diagnostic ultrasound instrument operating in combined pulsed and colour Doppler mode (Tarantal and Canfield 1993). Presentations at the 1993 Annual Conference for Ultrasonics in Biophysics and Bioengineering reported similar lesions in rabbits (O'Brien) and pigs, and the threshold for extravasation in mice lungs was less than 0.5 MPa (Carstensen). The mechanism is not fully understood, but is considered to be nontherreal and cavitation-related. Cavitation index

It is intended that a biologically relevant cavitation "index" based on physical parameters be pro-

Biophysical effects of ultrasound • S. B. BARr,'ET'ret al.

duced to predict the likelihood of adverse consequences (biological or physical) of an ultrasonic exposure. The use of biophysical endpoints is being considered by the International Electrotechnical Commission (IEC) Technical Committee 87. Meanwhile, a different system has been adopted in the USA (AIUM/ NEMA 1992) with the development of a predictor of cavitation known as the "Mechanical Index." Values are now being given in some literature in terms of MI. This new parameter is given by the expression MI = P.3(Zsp)/ff~ where Pr.3(Zsp) is the peak rarefactional pressure (in MPa) derated by 0.3 dB/cm.MHz to the point on the beam axis (Zsp), where the pulse intensity integral (PIL3) is maximum, and fc is the centre frequency (in MHz). The MI was developed to attempt to predict collapse cavitation events which, from a bioeffects aspect, may be the most severe. From a clinical perspective, the equipment output display of MI is intended to predict the likelihood of cavitation occurring in each examination mode. However, the model does not consider dwell time (which is related to operator skill and examination difficulty) or patient temperature (which may affect gas tension and bubble development). The MI does not address the issues of stable cavitation, or any other nonthermal phenomena that may be dependent on the pulse length or the duration of the exposure. The development and definition of the MI as a predictor of cavitation in vivo relied on data from specialised in vitro studies that used controlled nuclei environments to model bubble dynamics. It demonstrated that under idealised experimental conditions (liquid containing polystyrene microspheres as stabilised bubbles of resonant size), cavitation can be produced under pulsing conditions equivalent to that emitted by diagnostic equipment (Apfel and Holland 1991; Holland et al. 1992). This research focused specifically on transient cavitation phenomena, with the underlying assumption that bubbles of all radii are present in an exposed medium (Apfel 1986; Holland and Apfel 1989). It takes no account of effects, such as the growth of bubbles by rectified diffusion. It was suggested that MI values of less than 0.5 will not lead to phenomena associated with collapse cavitation (Crum et al. 1992; Holland and Apfel 1990). The MI does not take account of pulsing parameters, such as pulse length and duty cycle. In fact, there is some evidence to suggest that the pressure, Pr, threshold for cavitation detection is lowered with increasingly higher duty cycles (Calabrese et al. 1993). Pulse length has been shown to affect the cavitation

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threshold for specialised in vitro experimental conditions when sonochemiluminescence was used as the detection method (Fowlkes and Crum 1988). However, pulse length had no effect on the threshold when the system used polystyrene microspheres and a passive acoustic detector (Calabrese et al. 1993). While the concept of output display systems is gaining support, the process of achieving that aim is not universally accepted. The AIUM/NEMA mechanical index is computed from a value of the applied Pr that is derated on the basis of an assumed attenuation factor. This is, at best, a rough approximation due to the difficulties of modeling the complex acoustic and biological properties of human tissue, and the difficulties of covering widely varying exposimetry in sophisticated modern autoscanning systems. The large number of complicated-looking equations given in the documentation imply that the indices provide precise predictions of in situ exposures under all conditions. In fact, the models have yet to be thoroughly validated. The use of derated acoustical exposure values also does not allow the worst-case conditions to be displayed. The IEC (Technical Committee 87: Ultrasonics) is currently developing an international standard to classify equipment in biophysical terms (ter Haar et al. 1989) based on its ability to cause cavitation in human tissues in vivo (Preston 1992). The IEC is taking a cautious approach and favours classifying equipment according to its acoustic field calibrated in water, rather than using a derated value that may be difficult to trace. Due to the inadequate empirical data forming the basis of these output displays, there are some differences between AIUM/NEMA and IEC on fundamental issues. For instance, it is not yet resolved whether or not cavitation events are frequency dependent. The IEC index is still in draft stages of preparation but is intended to classify equipment according to its acoustic field. Class A equipment may be used without reservations. As modern ultrasonographic devices use large numbers of dynamically changing exposure conditions during a clinical examination, the likelihood is that most equipment will be grouped into class B. The difficulty will then be to alert the operator to the relative risk from different exam modes using the same instrument. The dependence of cavitation on tissue properties is illustrated by the results of studies with lithotripter and ultrasound exposures on mice. When the kidney was insonated with an lsppA intensity of 140 W/cm2, or Pr pressure of 5.0 MPa, and a frequency 4 MHz (in the range of imaging frequencies), virtually no bioeffects were seen (Carstensen et al. 1990). It was estimated that MI value was 1.2 at the focal point. However, in another study in which the mouse lung was

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insonated with an ISPPA intensity of 180 W/cm2, the onset of cavitation correlated with the calculated value of the MI, and the threshold was found to be MI = 0.5 (Child et al. 1990). The effect was absent in fetal lung, indicating that the presence of nuclei or microscopic air bubbles (or air spaces, as exist in the lungs) is crucial for the occurrence of collapse cavitation-type of bioeffects in vivo. A number of papers report soft tissue damage resulting from exposure of experimental mammals to fields of extracorporeal shock wave lithotripters (Delius et al. 1987, 1988). Thresholds have been determined for biological effects in various tissues, with the lowest threshold of 1-2 MPa occurring in lung tissue (Hartman et al. 1990). Extravasation in kidney tissue has been reported at peak rarefaction pressures of 3 5 MPa (Mayer et al. 1990), which is within the range reported for diagnostic Ultrasound exposures (Duck 1989). While there is a need for better understanding of the differences between diagnostic ultrasound and lithotripsy exposures, it is evident that the mouse lung is damaged by exposures to pulsed ultrasound and lithotripters at similar threshold levels. As the calculated MI value is derated as a function of both increasing frequency and increasing distance from the transducer, it is possible that two different ultrasound imaging devices may have the same MI value, but different IsppA values. Due to the variability in the composition and properties of the intervening tissues, it is uncertain whether these devices are substantially equivalent in terms of risk of cavitational bioeffects. In summary, despite its limitations the MI provides some indication of clinically relevant bioeffects produced by inertial cavitation. However, its use in predicting other nonthermal bioeffects is not obvious. The World Federation for Ultrasound in Medicine and Biology has made a commitment to sponsor a symposium on safety of medical ultrasound, in 1994, to determine the international consensus on issues regarding ultrasonically induced cavitation and other nonthermal interactions. OTHER N O N T H E R M A L M E C H A N I S M S If a diagnostic ultrasonic exposure is localised to a region of the order of a wavelength in size, bulk heating effects are minimal and nonthermal effects may become the dominant interactive mechanism between ultrasound and tissue. Stresses in biological media result from physical forces associated with radiation pressure and torque, radiation forces, and acoustic streaming (Nyborg 1978). In particular, acoustic streaming near boundaries can lead to localised cell membrane rupture or cell lysis.

Volume20, Number3, 1994 Radiation pressure Radiation pressure is exerted on any body immersed in an acoustic field. In a standing wave field, the radiation pressure is greatest at pressure maxima and least at velocity maxima. Changes in the auditory response in cats during exposure to pulsed ultrasound have been attributed to the interaction of radiation pressure with the brain (Foster and Wiederhold 1978). Similarly, focused ultrasound exposure of the brain has produced changes in hearing (Gavrilov et al. 1976). Radiation force Radiation force is exerted on any body immersed in the acoustic field. In a plane-traveling wave field, the force is in the direction of propagation and is proportional to intensity. It also depends on the properties of the object, in particular its size and density (fractional density difference) in comparison with that of the surrounding medium. In a standing wave field, the situation is complex and the direction of the force on the object depends on its properties. Dyson et al. (1974) observed red cell aggregations into bands within the capillaries of chick embryos. Band separation corresponded to a distance of one half wavelength and was produced with CW exposures at spatial average intensities of approximately 1 W/cm 2. Banding was (generally) reversible, while some irreversible damage to the epithelium was reported and attributed to cavitation. In many cases, second-order phenomena manifest themselves as platelet aggregation. Nyborg (1989) examined the criterion for aggregation of spherical particles and derived expressions for the critical intensity for particle aggregation with fractional density and particle radii as parameters. He developed a theory indicating that when an ultrasound beam produces an aggregation of small particles such as biological cells, the ISpTAintensity is the governing factor. For human platelets in plasma, the critical intensity level for aggregate formation was determined to be of the order of 100 mW/cm2. However, the clinical implications of these theoretical predictions are, as yet, unknown. It is worth noting that in commercial diagnostic equipment, typical IsvrA values (in water) range from 0.5 to 1.0 W/cmz for Doppler equipment, and values as high as 5.0 W/cmz have been reported (Ide 1989; Duck 1989; Zagzebski 1989). Agglomeration of particles in suspension around a gaseous body of diameter approximately 4 #m was reported at a CW exposure at 2.1 MHz and spatial peak intensity levels of 16-32 mW/ cm z, and were also produced by a 10 min exposure from a diagnostic Doppler instrument (Miller et al. 1979). Platelet aggregation was also observed during exposure to 30/zs pulses at 4 MHz with a pulse repetition rate of 30 Hz and Isr,rA levels as low as 30 mW/

Biophysical effectsof ultrasound • S. B. BARNE'rret al. cm 2 (Barnett and Kossoff 1984). Stabilized microbubbles were deliberately introduced into the field by means of a hydrophobic filter membrane. Acoustic torque

Nonuniformity of the acoustic field also leads to a time-independent twisting action which is referred to as acoustic torque. This torque produces a rotating movement of cells and intracellular structures in the medium and may result in a spinning motion of the cells in suspension (e.g., blood), or of intracellular structures (Nyborg 1978). Acoustic streaming

Acoustic streaming describes the movement of fluid in an ultrasonic pressure field (Nyborg 1965). Where fluid motion encounters boundaries, high velocity gradients can develop and produce substantial shear stresses. This may have biologically significant consequences if the boundary is formed by embryonic epithelia. Fluid motion around microscopic cavitation bubbles is known as acoustic microstreaming and shear stresses developed at cell membranes may be responsible for changes in membrane permeability. For microscopic streaming near ultrasonically resonating bubbles, it has been calculated (Nyborg 1978) that the threshold for bioeffects would be as low as 1 mW/cm 2 at 1 MHz frequency. Bulk, unidirectional, streaming in water, measured with hot film anenometry, has been reported at diagnostic intensities in pulsed, focused fields at 3.5 MHz frequency (Starritt et al. 1988). Streaming velocities increased with an increasing level of finite amplitude distortion in the acoustic wave generated by a commercial pulsed Doppler imaging device. It has been suggested that the enhanced streaming was due to the increased absorption of the distorted waveform. In the Doppler field, the maximum velocity measured was approximately 14 cm/s while in the imaging field an order of magnitude lower velocity of about 1 cm/s was observed. The implications of these findings for the safety of diagnostic ultrasound are yet to be established. Acoustic absorption in tissue is higher than in water (where bulk streaming experiments have been carried out) and this will significantly decrease the level of finite amplitude distortion in the propagating wave. Streaming has been shown to be an essential component in the transfer of energy that results in the disintegration of calculi by shock waves in lithotripter treatment of kidney stones (Holmer et al. 1991). Acoustic microstreaming is especially pronounced during the stable cavitation activity of gas bubbles. High velocity gradients and hydrodynamic shear stresses associated with microstreaming may lead

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to cell membrane breakage and haemolysis if a critical shear stress is exceeded. Theoretical predictions, involving gaseous body excitation in blood, indicate that CW excitation at a modest pressure amplitude of 0.02 MPa is sufficient to generate a force that leads to the critical stress (Lewin and Bjorno 1982; Nyborg 1978). These predictions agree with the experimental results (Miller 1987) of vigorous vibration of minute gas volumes in CW fields at spatial peak exposures as low as 10 mW/cm 2. Release of adenosine triphosphate (ATP) from red cells was reported (Miller 1987) both under CW (2 MHz, 5.6-100 mW/cm 2 spatial average intensity) and pulsed wave conditions (1 /zs pulses, 0.10.01 duty factor, spatial average intensity, 0.064-1.0 W/cm2). Structural changes in mammalian liver have been reported following exposure to 3 MHz pulses of 100 ms duration and intensity (most likely.-/srrP) 3000 W/cm 2 (Chan and Frizzell 1977). SUMMARY Thresholds for biological responses are (a) timedependent for thermal effects and (b) time-independent for cavitation/mechanical effects. However, the magnitude of the resulting biological effect is dependent on the duration of exposure for any mechanism once the threshold value has been exceeded. Thermal mechanism

The body temperature of humans is maintained within a relatively narrow range, and if exceeded, numerous adverse effects, including death, can result. Temperatures above 39°C reduce the mitotic activity of mammalian cells in culture. In humans, an elevated core temperature to 40.5°C induces reversible organ reactions in response to stress. Local temperatures above 43°C induce irreversible tissue damage. Hyperthermia is a teratogen in many animal species including nonhuman primates and humans. No adverse effects due to hyperthermia have been reported from temperature elevations less than 1.5°C above normal body temperature, even for prolonged exposures. Serious embryonic and fetal damage can result from temperatures above 41°C when maintained for 15 min or longer. The initiation of an adverse effect is directly proportional to the magnitude of the temperature elevation and to the logarithm of the exposure duration. The effects of elevated temperature are most dramatic at specific stages of embryonic development. For gross abnormalities in the CNS the susceptible stage is prior to organogenesis at the time of closure of the neural tube. In fetal development, interference with neurone migration (in the 2nd trimester in humans) may produce subtle neurological cognitive deficiencies that would be extremely difficult to detect.

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Cavitation mechanism

Cavitation-related events are believed to be responsible for most biological effects on in vitro cell suspensions, the most likely outcome being cell death. The death of fruit fly larvae exposed to diagnostic ultrasound indicates the potential for destructive cavitation with this modality, given a rich source of stabilised cavitation nuclei. Significant biological effects, associated with gas body activation, have been produced experimentally in mammalian lung tissue in different species including nonhuman primates. The effects were observed at combinations of pressures and pulse durations similar to those presently available in diagnostic ultrasound equipment, particularly as used in pulsed Doppler mode, The exact nature of the interactive mechanism is not fully understood, at present. Transient cavitation has been detected in aqueous solutions seeded with air bubbles and insonated with microsecond pulses at a peak rarefactional pressure, Pr, less than 1.0 MPa. However, there is no evidence that flowing cardiovascular blood supports cavitational activity in vivo. Other nonthermal mechanisms

There is some evidence that acoustic output levels similar to those produced by diagnostic ultrasound equipment can lead to significant biological effects in vitro, providing the gas pockets of appropriate size are present to generate fluid currents. There is some evidence of the existence of microbubbles in biological tissue. No immediate evidence exists that microstreaming leads to any significant adverse bioeffects at exposure conditions used in clinical diagnostic practice. However, it is possible that the extravasation of blood in lung tissue may result from the stresses induced by acoustic microstreaming within the surfactant or pleural layers. Second-order phenomena require further research, in particular experiments aimed at quantifying the critical stresses for membrane rupture, and the pressure amplitudes and frequencies for cavitation-like phenomena in mammalian tissues. Acknowledgements--The authors wish to thank members of Working Group 12 of the International Electrotechnical Commission Technical Committee 87, Ultrasonics, for their contributions during the formative stages of the preparation of this document. Valuable individual contributions were also received from Drs. D. Bacon, S. Bly, C. Burr, P. Carson, P. Edmonds, M. Edwards, J. Herbertz, G. Kossoff, P. Lewin, M. Miller, A. Mortimer, W. O'Brien, R. Preston and H.-Dieter Rott.

REFERENCES AIUM/NEMA. Standard for real-time display of thermal and mechanical acoustic output indices on diagnostic ultrasound equip-

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