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Heating of guinea-pig fetal brain during exposure to pulsed ultrasound
Ultrasound in Med. & BioL Vol. 19, No. 5, pp. 415-424, 1993
0301-5629/93 $6.00+ .00 © 1993 PergamonPressLtd.
Printed in the USA
OOriginal Contribution HEATING OF GUINEA-PIG FETAL BRAIN DURING E X P O S U R E TO P U L S E D U L T R A S O U N D K. L. BOSWARD, t S. B. BARNETT, ¢ A. K. W . WOOD, t M . J. EDWARDS t a n d G. KOSSOFF* *Department of Veterinary Clinical Sciences, University of Sydney, NSW, 2006, Australia, and *Ultrasonics Laboratory, CSIRO, Division of Radiophysics, 126 Greville Street, Chatswood, NSW, 2067, Australia (Received 25 September 1992; in final form 25 January 1993) Abstract--Ultrasound-induced temperature elevations in fresh and formalin-fixed fetal guinea-pig brains were measured during in vitro insonation, with a stationary beam in a tank containing water at 38°C. The pulsing regimen used 6.25 tzs pulses, repeated at a frequency of 4 kHz emitted from a focussed transducer operating with a centre frequency of 3.2 MHz. The greatest temperature rise in brain tissue occurred close to bone and correlated with both gestational age and progression in bone development. After a 2 min insonation with a spatial peak temporal average intensity (Isrrx) of 2.9 W/cm 2, a mean temperature elevation of 5.2°C was recorded in fetuses of 60 days gestation (dg). The same exposure produced an increase of 2.6°C in the centre of whole brains of 60 dg fetuses when the bony cranium was removed. As most of the heating occurs within 40 s, these findings have implications for the safety of pulsed Doppler examinations where dwell-time may be an important factor.
Key Words: Acoustics, Biological effects, Pulsed ultrasound, Temperature increase, Tissue heating, Ultrasound bioeffects, Ultrasound safety.
Temperature increase has been implicated as the cause of a range of developmental effects following experimental intrauterine exposures to ultrasound (Carstensen and Gates 1985). Fry (1986) reported that ultrasound-induced abnormalities in mouse fetuses occurred when the resting body temperature rose by 2.5 to 3.0°C. Fetal brain malformations, such as exencephaly, have been reported in mice following in utero insonation with continuous-wave (CW) ultrasound at a spatial average, temporal average intensity (IsATA) of 40 mW/cm 2 (Shoji et al. 1975), or with pulsed ultrasound at 586 mW/cm 2 (Takabayashi et al. 1985). Neither of these results have been reproduced, and careful attempts to duplicate the findings of the latter study have been unsuccessful (Child et al. 1984, 1988). Fetal brain abnormalities, including exencephaly, have been reported by Sikov and Hildebrand (1976) following exposure of pregnant rats, on day nine of gestation, to CW ultrasound at 10.5 W/cm 2 /SARAfor 5 to 15 min. It has also been clearly demonstrated in rodents that major fetal brain abnormalities follow systemic maternal hyperthermia of 2.5°C above normal body temperature (Edwards 1986, 1988). Similarly, Hendrickx et al. (1979) showed that raising the core temperature of pregnant monkeys
INTRODUCTION
The use of simple, real-time B-mode ultrasonic imaging in obstetrical examinations is generally considered safe (Wells 1987), given the absence of verified reports of adverse effects on mammalian tissues in more than three decades of clinical use (Ziskin and Pettiti 1988). The low power outputs used in real-time ultrasonic equipment, combined with the brief dwelltime of the beam, provide little opportunity for significant ultrasound-induced temperature increase in tissues during clinical imaging examinations (NCRP 1992; WFUMB 1992). However, currently available equipment with pulsed Doppler capability uses substantially higher power levels (Duck 1990; Duck et al. 1987; Duck and Martin 1991) to allow the detection of signals from small vessels in deep tissue. Furthermore, in a Doppler examination, the ultrasonic beam may be applied to a fixed volume of tissue for extended periods of time. As a consequence, such exposures have the potential to increase significantly the temperature of the tissues being examined. Address correspondence to: Ms. K. L. Bosward, C/-Ultxasonics Laboratory, CSIRO, Division of Radiophysics, 126 Greville Street, Chatswood, NSW, 2067, Australia. 415
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(Macaca radiata) by 2.4°C for 1 h on gestational days 27 to 30 produced a range of fetal malformations. A comprehensive review of the literature has demonstrated that central nervous system tissue is particularly sensitive to temperature elevations (Miller and Ziskin 1989). Due to its higher absorption coefficient for ultrasound, bone is heated during insonation to a greater extent than soft tissue. It is to be expected that tissues adjacent to, or encased in, bone may be significantly heated during pulsed Doppler ultrasonic examinations. Thus, the aim of the present study was to: I. Quantify the extent of temperature increase in the brain of fetal guinea-pigs during exposure to pulsed ultrasound, at intensities and pulsing regimes similar to those available for use during clinical pulsed Doppler examinations. 2. Examine the effect of gestational age and corresponding bone development on the extent and rate of heating of fetal guinea-pig brains during exposure to pulsed ultrasound.
Volume19, Number 5, 1993 The cross-axis intensity profile of the free-field ultrasonic beam in water was plotted in 0.1 mm steps using a 1.0 mm diameter needle-mounted polyvinylidine difluoride (PVDF) hydrophone (NTR Systems Inc., Seattle, USA). Micromanipulators positioned the hydrophone and transducer in a glass tank filled with distilled water and maintained at 38 °C. The profiles were constructed at two specific distances from the transducer (4.4 and 6.0 cm) where the maximum temperature elevation was recorded in tissue for each of the two power levels (260 mW and 1120 mW) used in these experiments (Fig. 1). The spatial peak pulse average (IsPPA) and spatial peak temporal average (IsPTA) intensities were computed from the hydrophone response using a waveform analyser (Data 6100, Data Precision Corp., Danvers, USA). At 6.0 cm distance from the transducer face, the values were 112 W/cm: /SPPAand 2.9 W/cm 2 ISPTA"The - 6 dB beamwidth was 0.27 cm. The average power, measured with a radiometer (Model UPM-DT-l, Ohmic
MATERIALS AND METHODS
~
580
Guinea-pigs (Cavia species) were time-mated to produce fetuses of specific gestational ages. Females were mated immediately following the onset of oestrus, day zero, which was detected by the opening of the vaginal membrane. Twenty-four hours after the onset of oestrus, the female was separated from the male. Pregnancy was diagnosed by the failure to return to oestrus after 16 days and by palpation of the amniotic vesicles at 19 days postoestrus. Pregnant guinea-pigs at 30, 40, 50 and 60 days gestation (dg) were euthanased by an intraperitoneal injection of pentobarbitone sodium (Euthatal 350, May and Baker Australia), and the fetuses were dissected from the uterus. Each fetus was weighed and identified. One fetus from each litter was chosen at random for use as a fresh experimental specimen, while the litter mates were fixed in 10% formalin for at least one week before being used in experiments.
Ultrasonic exposimetry A single lead zirconate titanate transducer with a diameter of 19 mm, radius of curvature of 100 mm, and centre frequency of 3.2 MHz was used in all experiments. The transducer was driven by a pulse function generator (Model 8116A, Hewlett-Packard, USA) and its output varied by means of a regulated power supply and custom-built power amplifier. Pulsing parameters similar to those used in some clinical pulsed Doppler examinations were chosen (pulse duration = 6.25 #s, pulse repetition frequency = 4 kHZ).
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Fig. 1. Cross-axis intensity profiles of the ultrasonic beam measured at distances of 4.4 and 6.0 cm from the transducer for power levels of 260 mW (upper) and 1120 mW (lower). Solid line curves show the profiles at the positions in the beam where maximum heating was recorded for each power.
Heating of guinea-pig fetal brain @ K. L. BOSWARDel al.
Instruments Inc., MD, USA) at the same axial distance was 260 mW. At the maximum power output used, 1120 mW, the greatest temperature increase was measured at a distance of 4.4 cm. Hydrophone measurements at this location yielded intensities of 91.5 W/cm 2 IsppA and 2.5 W/cm 2 IsvrA and a - 6 dB beamwidth of 0.72 cm.
Experimental procedure Each fetus was secured with surgical tape (Blenderm, 3M, MN, USA) to an acrylic framework over which a sonolucent membrane of surgical film (Vidrape, Parke-Davis, USA) was stretched. The relative positions of the transducer and the fetus were adjusted by micromanipulators and the distance between them was determined by measuring the pulse time delay with a hydrophone and oscilloscope (Model 465B, Tektronix, OR, USA). Temperature was measured using copper-constantan thermocoupies in 26 gauge (0.46 mm) stainless-steel needles (Physitemp Inc, N J, USA) inserted into the fetal tissue and connected to a digital thermometer (BAT 10, Physitemp Inc). Needle mounted thermocouples, rather than bare wires, were chosen to ensure repeatable positioning against the inner aspect of the skull bone. Constant depth of insertion was also reliably achieved. This improved accuracy of positioning was traded against the small risk of error due to viscous heating artifact in measurements made after ultrasound had passed through bone. The absorption of sound in bone is so high as to minimise the effect of localised artifactual heating. Accurate positioning of each thermocouple in the beam was ensured by maximizing the temperature response to an initial lowlevel insonation. The tissue temperature was then allowed to equilibrate to 38°C before recording temperatures at 10 s intervals throughout the 120 s ultrasonic exposure and subsequent cooling periods. In three separate studies, measurements were taken from five fetuses at each gestational age and for each exposure condition. The mean peak temperature levels were calculated from the raw data and recorded in the results. The three studies were as follows.
Single point temperature measurement in fresh and fixed fetal brains. A single thermocouple was inserted to a depth of 0.3 cm in the brain tissue directly under the occipital bone. The ultrasound beam was applied to intercept the brain in the midsagittal plane. The purpose of this experiment was to determine the maximum increase in temperature that occurred in the region of proliferating neural tissue close to bone. Results from fresh and formalin-fixed brains were compared to determine whether the temperature increase was altered significantly by tissue fixation.
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Multiple temperature measurements in fixed fetal brains. Three thermocouples were inserted, in a transverse plane, through the skull to a depth of 0.3 cm. The tip of the thermocouple closest to the transducer lay in the brain tissue directly under the left parietal bone of the skull. The second thermocouple lay between the cerebral hemispheres, while the third was positioned under the right parietal bone. The aim was to examine further the effect of proximity to bone and the role of the brain in attenuating the ultrasonic beam.
Single point temperature measurement in isolated fixed fetal brains. After removing the brain from the braincase, a single thermocouple was inserted into the midcerebral region. This was done to determine the extent of heating due to absorption by the soft tissue without influence from nearby bone. The mean temperature increases were recorded from six formalin-fixed brains of 60 days gestational age guinea-pig fetuses.
Assessment offetal bone development To obtain a qualitative assessment of the extent of ossification, five fresh fetuses from each gestational age were radiographed in lateral recumbency, using a plastic cassette with a single intensifying screen (KMR, Eastman-Kodak, Rochester, USA) and an autochromatic film (Min R, Eastman-Kodak). The xray equipment (Faxitron 43805N, Hewlett-Packard, Germany) was designed to give high-resolution images of small thicknesses of tissue. In addition, a quantitative measure of the bone mineral content was obtained using an ash determination technique. The fetal skull was removed from the body at the atlanto-occipital joint, homogenized, placed into a preweighed ceramic crucible (Infusil, Bhanu Scientific Instruments Co., Bangalore, India) and dried overnight in an oven at I00-150°C. The crucibles were cooled in a dessicator and the dry-matter weight calculated before being heated to 600°C for 2 h in a muffle furnace (Martin Furnace and Engineering Pty. Ltd., Sydney, Australia). The ash, or total mineral content of the skull, was determined for each fetus and expressed as a percentage of total dry weight.
Statistical analyses All statistical analyses of variance were carried out using the Genstat 4.03e statistical package (1980 Lawes Agricultural Trust, Rothamstaed Experimental Station). The ash determination results were analysed as a completely random design, while all the other studies were analysed as completely random design split-plots. In the studies of heating in fresh versus fixed brains, the whole plots were gestational ages,
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and the different power levels were treated as subplots. In the study of multiple temperature measurements in fixed brains, the whole plots were again the different gestational ages, but the subplots were factorial, including both the different power levels and the different thermocouples. RESULTS
Single point temperature measurement in fetal brains Fresh brains. For each gestational age, a rapid temperature increase was recorded over the initial 40 s, followed by a plateau, and then rapid cooling when the ultrasound exposure ceased. At a power output of 260 mW (2.9 W/cm 2 ISVrA), the position of the beam focus (determined by plotting the - 6 dB beam profile) was 6.7 cm from the transducer, but maximum tissue heating was obtained at a 6.0 cm distance. For a power output of 1120 mW (2.5 W/cm 2 ISVrA), the focal distance was 6.0 cm, but the maximum tissue temperature was recorded at a distance of 4.4 cm. At these distances, the mean peak temperature increases for 260 mW and 1120 mW respectively ranged from 1.3 ° and 4.5°C at 30 dg to 4.7°C and 14.9°C at 60 dg (Table 1). Analysis of variance of the peak brain temperatures revealed significant differences related to the age of gestation (p < 0.01) and ultrasonic power
Table 1. Mean peak temperature increases in fresh and formalin-fixed fetal guinea-pig brains after 120 s exposure to pulsed ultrasound. Mean peak temperature increase
(°C) Gestational age (days) Fresh brains 30 30 40 40 50 50 60 60 Fixed brains 30 30 40 40 50 50 60 60
BPD (cm)
Distance (cm)
260 mW
1120 mW
0.7
4.4 6.0 4.4 6.0 4.4 6.0 4.4 6.0
0.9 1.3 1.1 1.3 2.7 3.5 3.2 4.7
4.5 4.6 4.9 4.2 11.1 10.1 14.9 14.3
1.5 2.0 2.5 0.7 1.5 2.0 2.5
4.4 6.0 4.4 6.0 4.4 6.0 4.4 6.0
4.9 1.4 5.0 1.5 10.8 3.5 13.4 4.2
BPD = biparietal diameter; distance = from transducer to thermocouple in brain under the occipital bone.
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level (p < 0.01). The least significant difference (l.s.d.) between the mean peak temperatures for each gestational age, averaged over the two power levels, revealed that the means for the 30 and 40 dg fetuses were significantly lower (p < 0.05) than those for the 50 and 60 dg fetuses. Whereas the means for the 30 and 40 dg fetuses were not significantly different from each other, the difference between the means of the 50 and 60 dg fetuses was significant (p < 0.05).
Fixed brains. The mean peak temperature for 260 mW and 1120 mW exposures ranged from 1.4 ° and 4.9°C, for fetuses of 30 dg, to 4.2 ° and 14.1 °C for those of 60 dg (Table 1). As in the fresh brains, a rapid rise in temperature was followed by a plateau, and rapid cooling when insonation ceased (Fig. 2). The shape of the plateau phase of the heating curve is illustrated in Fig. 3 which shows results of 3 min exposures to 1120 mW power. There is minimal increase in the mean temperature levels beyond the 2 min exposure duration. For ease of correlation, all data shown in the tables of results represent mean values obtained after exposure durations of 2 min. Analysis of variance of the peak temperatures revealed significant differences related to gestational age (p < 0.01) and ultrasonic power level (p < 0.01). The 1.s.d. between the means for each gestational age, averaged over the two power levels, revealed that the means for the 30 and 40 dg fetuses were significantly lower than those for the 50 and 60 dg fetuses (p < 0.05). Analysis of variance of the peak temperature in fixed and fresh fetal brains revealed no significant difference between the means at either power level or gestational age. Multiple temperature measurements in fixed fetal brains The highest temperature was consistently recorded from the thermocouple located in the brain tissue under the parietal bone closest to the transducer (Table 2, thermocouple 1). The mean peak temperature increases after 120 s exposure to 260 mW ranged from 1.2°C at 30 days to 5.2°C at 60 days gestational (dg). The respective values for exposure to 1120 mW were 3.4 ° and 15.9°C. The largest temperature increases were always observed in 60 dg fetuses. Measurements from the thermocouples in the midcerebral region yielded temperature increases at the same gestational ages ranging from 0.6 ° to 0.8°C at 260 mW, and 2.5 ° to 3.4°C at 1120 mW power. In this situation, the greatest temperature increase was measured in 50 dg fetuses (Table 2), where the mean peak temperatures for 260 mW and 1120 mW power outputs were 0.9 ° and 4.4°C, respectively.
Heating of guinea-pig fetal brain • K. L. BOSWARDel
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Similarly, results from the thermocouple placed under the distal parietal bone o f the skull showed the largest temperature increases to occur in 50 dg fetuses, ranging from 1.5 ° to 5.6°C for 260 m W and 1120 m W , respectively (Table 2). T h e smallest t e m p e r a t u r e increase (0.9°C) was recorded in the distal thermocouple in 60 dg fetuses exposed to 260 m W power output. T e m p e r a t u r e s measured from thermocouples close to the proximal (Fig. 4) and distal parietal bones
rose rapidly at both power levels over the initial 40 s, followed by a plateau, and rapid cooling when the ultrasound exposure ceased. The temperature in the midbrain increased at a slower rate without reaching the same plateau region, and cooling was slower. Analysis o f variance revealed a significant difference between the m e a n peak temperatures at different power levels for all gestational ages (p < 0.05 at 30 and 40 d g ; p < 0.01 at 50 and 60 dg). The l.s.d, o f the m e a n peak temperatures showed that at 30, 50 a n d 60
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Volume 19, Number 5, 1993
Table 2. M e a n peak temperature increases in fixed fetal guinea-pig brains after 120 s exposure to pulsed ultrasound in the transverse plane. Mean peak temperature increase (°C) Gestational age (days)
Bone thickness (mm)
Axial distance (cm)
th. 1
th. 2
th. 3
0.17 0.23 0.30
6.0 6.0 6.0 6.0
1.2 1.6 3.7 5.2
0.6 0.9 0.9 0.8
1.0 1.1 1.5 0.9
Power = 260 mW 30 40 50 60 Isolated brain 60 Power = 1120 mW 30 40 50 60 Isolated brain 60
6.0 0.17 0.23 0.30
4.4 4.4 4.4 4.4
0.00
6.0
2.6 3.4 4.7 11.7 15.9
2.5 3.9 4.4 3.4
3.1 4.3 5.6 3.6
7.7
th. 1 = thermocouple positioned under the proximal parietal bone; th. 2 = middle of cerebral hemispheres; th. 3 = under the distal parietal bone; distance = transducer to thermocouple.
Assessment offetal bone development
dg, the increase at the first thermocouple was significantly different (p < 0.05) from that at the second and third thermocouples.
Fetal radiography. Radiographs of the 30 dg fetuses showed the beginnings of ossification in the ribs, maxilla, mandible and the orbital area. At 40 dg, ossification had progressed to include the vertebrae, with further development of the radius, humerus, scapula, femur and tibia. The skull showed thickening of the bones of the braincase, orbit, mandible and maxilla. The tympanic bullae were prominent. By 50 dg, the appendicular skeleton had developed significantly
Insonation of isolatedfixed fetal brain A mean peak temperature increase of 2.6°C occurred in brains of 60 dg fetuses after insonation for 120 s (Fig. 5) at 260 mW power when positioned 6.0 cm from the transducer. When the power output was increased to 1120 mW, the mean peak temperature increase was 7.7°C (Table 2). 6¸ 5.
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60 days gestation
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50 days
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Fig. 4. Mean temperature elevations in fixed guinea-pig fetal brain tissue during 120 s exposures to 260 m W pulsed ultrasound. A single thermocouple was placed under the parietal bone at a distance o f 6.0 cm from the transducer.
Heating of guinea-pig fetal brain • K. L. BOSWARDet al.
421
10 9 1120 mW
Temperature
increase (°C)
0 0
60
~ 120
180 240 Time (secs)
300
360
Fig. 5. Mean temperature increases in the midcerebral region of 60 dg fetal guinea-pig brains isolated from the cranium and exposed to pulsed ultrasound at 260 mW and 1120 mW power.
and each vertebra was prominent. The teeth in both the mandible and maxilla were evident and the tympanic buUae were especially thickened. At 60 dg the skeleton was almost completely ossified. Fetal ash determination. The ash content of the skull increased significantly (p < 0.01) with gestational age (Fig. 6). The 1.s.d. showed that the ash content at each gestational age was significantly different from all the other ages (p < 0.05). DISCUSSION
ture change increased significantly with advancing gestational age. It is uncertain how much of the enhanced temperature increase was due to a larger area of bone absorbing more ultrasound. In the present study, the results of fetal ash assay demonstrated progressive increase in mineralisation consistent with increasing gestational age from day 30 onwards; it is known that ossification in the guineapig fetus begins on the 27-28th day (Graham and Scothorne 1970). The large difference in the maximum brain heating that occurred between 40 and 50 dg is apparently due to significant bone development
This study has demonstrated significant t e m p e r a ture increases in brain tissue adjacent to bone. Mean temperature increases of 4.7°C (occipital bone) and 5.2°C (parietal bone) were recorded in 60 dg fetuses, after 120 s exposure to an intensity of 2.9 W/cm 2 IsrrA (260 mW). A recent study reported a temperature increase of 5.6°C in the cranial bone in young mice exposed for 45 s to focussed pulsed ultrasound with an ISPTAof 1.5 W/cm 2 (Carstensen et al. 1990). Temperature measurements were made at the front surface of the 0.5 mm thick mouse skull bone, while our results were obtained at the bone/soft tissue interface inside the 0.3 mm thick guinea-pig skull bone. It is possible that our results may have been underestimated due to heat conduction along the thermocouple needle. In an in vitro study of heating in human fetal femurs (Drewniak et al. 1989), a temperature rise of 4.0°C was recorded in the diaphysis of a 108 dg femur after 60 s exposure to CW ultrasound at an IsrrA of 1.0 W / c m 2. In this latter study, the magnitude of tempera-
% ash
30
40
50
60
Gestational age (days) Fig. 6. Total ash content of guinea-pig fetal skulls expressed
as a percentage of dry skull weight, and plotted as a function of advancing gestational age.
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Ultrasound in Medicineand Biology
providing a larger volume of bone (increased skull diameter and thickness) for ultrasound absorption in the older fetuses. Fetal radiography provided qualitative confirmation of the progression in bone development and supported the data obtained from ash determination. During ultrasonic exposure, the pattern of brain heating was similar for insonations in the sagittal and transverse planes, with the highest temperatures generally being recorded in the older fetuses. The peak temperature elevations in the transverse approach resulted primarily from absorption of ultrasound by the parietal bones, with conduction of heat to the adjacent soft tissue. At each gestational age, the temperature increase in the middle of the cerebral hemispheres was lower than that recorded near bone. The slower rate of temperature rise suggests conduction of heat from the parietal bones in addition to local heating from the absorption of ultrasound by the brain tissue. The resultant heating of the central brain depends on the extent of bone formation and ultrasound attenuation. In late gestation (60 dg), thicker bone attenuates the incident ultrasound beam. This reduces the energy level available for absorption by soft tissue, while rapidly generating heat that can be conducted into the brain tissue. The effect of bone thickness on heating in equivalent volumes of brain tissue is demonstrated by the factor of three increases in soft tissue heating when the brain was insonated after removal from the cranium of 60 dg fetuses. The mean peak temperature in the brain tissue close to bone was found to increase with advancing gestational age and increased ultrasound exposure. The temperature increase was also a function of the distance of the specimen from the transducer, where maximum tissue heating occurred at an axial position closer to the transducer than the geometric focus of the beam. For a power output of 260 mW, the position of the beam focus was 6.7 cm from the transducer, but maximum tissue heating was obtained at a distance of 6.0 cm; for a power output of 1120 mW the focal distance was 6.0 cm, but the maximum tissue temperature was recorded at 4.4 cm. This finding is similar to a report of greater heating "in front of the focus" in soft tissue-mimicking gels (Bacon and Carstensen 1990) under conditions of nonlinear propagation. Furthermore, the present findings have questioned the relevance OflsrrA intensity as a predictor of tissue heating. In this study, the measured value of the IsP'rA (and ISPPA)was similar for power outputs of 260 mW and 1120 mW due to differences in beamwidth with distance from the transducer. However, heating was greater at 1120 mW where there was a wider
Volume19, Number 5, 1993 heated area. For the narrower beam at 260 mW and 6 cm distance, a greater thermal gradient would allow more rapid heat dissipation from the axis. The situation is further complicated by nonlinear processes which increase as a function of distance and power and provide opportunities for shock-loss to the medium. In in vitro studies, ter Haar et al. (1989) also reported that neither IsrrA nor /SARA showed good correlation with the temperature increase during a 1 min insonation of excised liver. The intensity values measured under free-field conditions in water are not the same as the in situ values that are subjected to attenuation from the tissue. In addition, the nonlinear distortion associated with higher peak positive pressure amplitudes leads to enhancement of the predicted temperature rise due to the absorption of the higher harmonic components (Swindell 1985). In the present study, the width of the ultrasound beam played a role in determining the temperature obtained in the tissue. The beam width measured at 6.0 cm axial distance for a power of 260 mW was 0.27 cm, while at 4.4 cm and 1120 mW it was 0.72 cm. The wider beam at 1120 mW means that the IsrrA is low but a larger area of tissue is heated. The effects of nonlinearity, with higher peak positive pressure amplitudes and the associated absorption of higher harmonic components, may also be responsible for the greater differences in the temperature increases recorded at the higher exposure levels. The shape of the graph of temperature increase versus time is asymptotic. Initially, the slope is steep with more than 75% of the temperature rise occurring in the initial 40 s when temperature increases linearly with time in proportion to the absorbed energy. This is followed by a slower rate of temperature rise as heat is conducted from the exposed area of tissue. In the final phase the curve flattens, representing the establishment of a thermal equilibrium as heat is dissipated. The effectiveness of the water tank (maintained at a constant temperature of 38°C) in dissipating heat is shown by the rapid decrease in brain temperature that occurs when the ultrasonic exposure ceased. This cooling results from thermal conduction, as a temperature gradient is established between the heated tissue and the water tank. Thermal conduction occurs as soon as a temperature gradient is established in a tissue, and the heat diffuses away in an attempt to eliminate the gradient. In situ cooling comes from two main sources: thermal conduction and tissue perfusion. Although equations for a cooling function have been proposed (WFUMB 1992), it is difficult to estimate the effect of perfusion on heat dissipation, since vascularity varies between and within tissues at any one time, depending on the organ and physiologi-
Heating of guinea-pig fetal brain • K. L. BOSWARDel al.
cal state. In vivo studies on the cooling effect of blood circulation in fetal tissues exposed to ultrasound have reported smaller temperature increases in perfused tissue than in avascular tissue (Abraham et al. 1989; Sikov et al. 1984). The effect of tissue perfusion and its role in heat dissipation, compared to the relatively large volume of temperature-stabilised water in a tank, requires further investigation. Few studies have determined the temperature rise in fetal tissue during exposure to ultrasound. Abraham et al. (1989) measured temperature increases in rat fetuses at 15-20 dg, and reported an increase of 10.0°C in fixed fetuses after exposures of l0 min to CW/SARA intensity of 4.2 W / c m 2. In live fetuses the same exposure gave temperature elevations of I 1.0 ° and 12.8°C with and without blood circulation, respectively. Sikov et al. (1984) measured the temperature in the exteriorised uterine horns of 9 dg rats. During 15 min exposures to CW ultrasound, at intensities of 1.8 to 20 W / c m 2, it was shown that ligation of ovarian and uterine arteries resulted in a greater temperature rise. During in vivo exposures of mouse embryos, prior to bone development, Fry (1986) observed greater heating from pulsed-wave (PW) than from CW ultrasound. Exposure for 20 s to an intensity of 27.0 W / c m 2 ISPTA gave a maximum temperature rise of 4. l °C with CW, and 8.0°C with PW ultrasound. The result was attributed to the highly nonlinear sound field conditions associated with the pulsing regime. The significant heating of fetal brain found in the present study occurred with ultrasonic pulsing conditions, power outputs and ISPTAvalues similar to the range of values reported for modem pulsed Doppler equipment (Duck et al. 1987; Duck 1989, 1990; Stewart et al. 1986). Although a higher average power output (l 120 mW) was used for the upper level, its average intensity (IsrrA) value, expressed over the larger beamwidth, was similar to that for 260 mW in the narrower beam. In the present in vitro study, the fetuses were dissected free from the uterus. In vivo, the matemai abdominal wall would attenuate ultrasonic energy and smaller elevations in the temperature of the central nervous system tissues might be expected. If, however, an acoustically transparent window, such as the bladder, were used during a pulsed Doppler ultrasonic obstetric examination, minimal loss of power would occur once the ultrasonic beam had passed through the maternal abdominal wall. Rather than attenuating, these fluids offer an opportunity for the development of a shock front that may enhance heating effects of pulsed ultrasound beams (Bacon 1984). Thus, there is a need for further in vivo observations to determine what, if any, effect the attenua-
423
tion of the ultrasonic beam by the maternal abdominal wail, or other overlying structures, has on temperature elevations in fetal tissues. The central nervous system is known to be especially sensitive to heat (Edwards 1986) and temperature elevations of 2 ° to 2.5°C are teratogenic to the developing nervous tissue in experimental animals (Edwards 1988). The nature of the biological effect depends on the duration of the hyperthermic exposure and the stage of pregnancy when it occurred. Reported defects include talipes, arthrogryposis, microphthalmia, micrencephaly, neural tube defects and other functional defects (Edwards 1988). Using an in vitro whole-embryo culture system, Barnett et al. (1990) exposed rat embryos at 9.5 dg to pulsed ultrasound and elevated temperature. Effects included decreased somite number, significant reductions in the ratios of brain:body area, and a characteristic oedematous-blistering of the prosencephalon, after exposure to ISPTA 1.2 W / c m 2 together with a 1.5°C temperature elevation for 15 min. A review of the literature on mammalian hyperthermia (Miller and Ziskin 1989) showed that the higher the temperature, or the longer the hyperthermic exposure, the greater the chance of damage occurring in mammalian tissue. Accordingly, an absolute temperature of 43.2°C, as observed in the present study following 2 min exposure to 2.9 W / c m 2/SPTA, would need to be maintained for 1 min for significant levels of damage to occur. Similarly, l0 min at 41.5°C would also be expected to cause tissue damage. The present findings were obtained under a specific set of experimental conditions which did not take account of attenuation by the maternal abdominal wall and cooling by vascular perfusion. It is unlikely that these conditions would be duplicated during routine pulsed Doppler clinical examinations. Ultrasonologists and ultrasonographers should be aware, however, of the possible damaging effects of hyperthermia. Care should be taken to minimize the dwell-time of the ultrasonic beam on the fetal tissue and to minimize the power outputs used with pulsed Doppler equipment in clinical examinations. Acknowledgement--The financial support of the Selby Scientific Foundation is gratefully acknowledged.
REFERENCES Abraham, V.; Ziskin, M. C.; Heyner, S. Temperature elevation in the rat fetus due to ultrasound exposure. Ultrasound Med. Biol. 15:443--449; 1989. Bacon, D. R. Finite amplitude distortion of the pulsed fieldsused in diagnostic ultrasound. Ultrasound Med. Biol. 10:189-195; 1984. Bacon, D. R.; Carstensen E. L. Increased heating by diagnostic ul-
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trasound due to nonlinear propagation. J. Acoust. Soc. Am. 88:26-34; 1990. Barnett, S. B.; Walsh, D. A.; Angles, J. A. Novel approach to evaluate the interaction of pulsed ultrasound with embryonic development. Ultrasonics 28:166-170; 1990. Carstensen, E. L.; Child, S. Z.; Norton, S.; Nyborg, W. L. Ultrasonic heating of the skull. J. Acoust. Soc. Am. 87:1310-1317; 1990. Carstensen, E. L.; Gates, A. H. Ultrasound and the fetus. In: Nyborg, W. L.; Ziskin, M. C., eds. Biological effects of ultrasound. Clinics in diagnostic ultrasound, Vol. 16. New York: Churchill Livingstone; 1985:85-95. Child, S. Z.; Carstensen, E. L.; Davis, H. A test for the effects of low temporal average intensity pulsed ultrasound on the rat fetus. Exp. Cell Biol. 52:207-210; 1984. Child, S. Z.; Carstensen, E. L.; Gates, A. H.; Hall, W. J. Testing for the teratogenicity of pulsed ultrasound in mice. Ultrasound Med. Biol. 14:493-498; 1988. Drewniak, J. L.; Carues, K. I.; Dunn, F. In vitro ultrasonic heating of fetal bone. J. Acoust. Soc. Am. 86:1254-1258; 1989. Duck, F. A. Output data from European studies. Ultrasound Med. Biol. 15 (Suppl. 1):61-64; 1989. Duck, F. A. Two case studies in ultrasound exposure measurements. Br. J. Radiol. 63:392; 1990. Duck, F. A.; Martin, K. Trends in diagnostic ultrasound exposures. Phys. Med. Biol. 15:1423-1432; 1991. Duck, F. A.; Starritt, H. C.; Anderson, S. P. A survey of the acoustic output of ultrasonic Doppler equipment. Clin. Phys. Physiol. Meas. 8:39-49; 1987. Edwards, M. J. Hyperthermia as a teratogen: A review of experimental studies and their clinical significance. Teratogenesis Carcinog. Mutagen. 6:563-582; 1986. Edwards, M. J. Hyperthermia and the developing central nervous system. Congen. Anomal. 28 (Suppl.):S119-S133; 1988. Fry, F. J. Effects of ultrasound exposure on pregnancy: A study in the mouse. IEEE Trans. Ultrason. Ferroelectr. Freq. Contr. 33:225-234; 1986. Graham, R. W.; Scothorne, R. J. Calcium homeostasis in the fetal guinea-pig. Q. J. Exp. Physiol. 55:44-53; 1970. Hendrickx, A. G.; Stone, G. W.; Henrickson, R. V.; Matayoshi, K.
Volume 19, Number 5, 1993 Teratogenic effects of hyperthermia in the bonnet monkey (Macaca radiata). Teratology 19:177-182; 1979. Miller, M. W.; Ziskin, M. C. Biological consequences ofhyperthermia. Ultrasound Med. Biol. 15:707-722; 1989. NCRP. Exposure criteria for medical diagnostic ultrasound: 1. Criteria based on thermal mechanisms. Report No. 113. Bethesda, MD: National Council for Radiation Protection and Measurements; 1992. Shoji, R.; Murakami, U.; Shimizu, T. Influence of low-intensity ultrasonic irradiation on prenatal development of two inbred mouse strains. Teratology 12:227; 1975. Sikov, M. R.; Collins, D. H.; Cart, D. B. Measurement of temperature rise in prenatal rats during exposure of the exteriorized uterus to ultrasound. IEEE Trans. Sonics Ultrason. SU-31:497503; 1984. Sikov, M. R.; Hildebrand, B. P. Effects of ultrasound on the prenatal development of the rat. Part 1.3.2 MHz continuous-wave at nine days of gestation. J. Clin. Ultrasound 4:357-363; 1976. Stewart, H. F.; Silvis, P. X.; Smith, S. W. Patient exposure data for Doppler studies. Clin. Diagn. Ultrasound 17:187-195; 1986. Swindell, W. A theoretical study of nonlinear effects with focused ultrasound in tissues: An acoustic bragg peak. Ultrasound Med. Biol. 11:121-30; 1985. Takabayashi, T.; Sato, S.; Sato, A.; Ozawa, N.; Sou, S.; Yajima, A.; Suzuki, M. Influence of pulse-wave ultrasonic irradiation on the prenatal development of mouse. Tohuku J. Exp. Med. 147:403-410; 1985. ter Haar, G. R.; Duck, F.; Daniels, F. A.; Starritt, H. Biophysical characterisation of diagnostic ultrasound equipment--Preliminary results. Phys. Med. Biol. 34:1533-1542; 1989. Wells, P. N. T. The safety of diagnostic ultrasound. Br. J. Radiol. (Suppl. 20). London: British Institute of Radiology; 1987. WFUMB. World Federation for Ultrasound in Medicine and Biology Symposium on Safety and Standardisation in Medical Ultrasound: Issues and recommendations regarding thermal mechanisms for biological effects of ultrasound. Barnett, S. B.; Kossoft, G., eds. Ultrasound Med. Biol. 18:(9); 1992. Ziskin, M. C.; Pettiti, D. B. Epidemiology of human exposure to ultrasound: A critical review. Ultrasound Med. Biol. 14:91-96; 1988.
0301-5629/93 $6.00+ .00 © 1993 PergamonPressLtd.
Printed in the USA
OOriginal Contribution HEATING OF GUINEA-PIG FETAL BRAIN DURING E X P O S U R E TO P U L S E D U L T R A S O U N D K. L. BOSWARD, t S. B. BARNETT, ¢ A. K. W . WOOD, t M . J. EDWARDS t a n d G. KOSSOFF* *Department of Veterinary Clinical Sciences, University of Sydney, NSW, 2006, Australia, and *Ultrasonics Laboratory, CSIRO, Division of Radiophysics, 126 Greville Street, Chatswood, NSW, 2067, Australia (Received 25 September 1992; in final form 25 January 1993) Abstract--Ultrasound-induced temperature elevations in fresh and formalin-fixed fetal guinea-pig brains were measured during in vitro insonation, with a stationary beam in a tank containing water at 38°C. The pulsing regimen used 6.25 tzs pulses, repeated at a frequency of 4 kHz emitted from a focussed transducer operating with a centre frequency of 3.2 MHz. The greatest temperature rise in brain tissue occurred close to bone and correlated with both gestational age and progression in bone development. After a 2 min insonation with a spatial peak temporal average intensity (Isrrx) of 2.9 W/cm 2, a mean temperature elevation of 5.2°C was recorded in fetuses of 60 days gestation (dg). The same exposure produced an increase of 2.6°C in the centre of whole brains of 60 dg fetuses when the bony cranium was removed. As most of the heating occurs within 40 s, these findings have implications for the safety of pulsed Doppler examinations where dwell-time may be an important factor.
Key Words: Acoustics, Biological effects, Pulsed ultrasound, Temperature increase, Tissue heating, Ultrasound bioeffects, Ultrasound safety.
Temperature increase has been implicated as the cause of a range of developmental effects following experimental intrauterine exposures to ultrasound (Carstensen and Gates 1985). Fry (1986) reported that ultrasound-induced abnormalities in mouse fetuses occurred when the resting body temperature rose by 2.5 to 3.0°C. Fetal brain malformations, such as exencephaly, have been reported in mice following in utero insonation with continuous-wave (CW) ultrasound at a spatial average, temporal average intensity (IsATA) of 40 mW/cm 2 (Shoji et al. 1975), or with pulsed ultrasound at 586 mW/cm 2 (Takabayashi et al. 1985). Neither of these results have been reproduced, and careful attempts to duplicate the findings of the latter study have been unsuccessful (Child et al. 1984, 1988). Fetal brain abnormalities, including exencephaly, have been reported by Sikov and Hildebrand (1976) following exposure of pregnant rats, on day nine of gestation, to CW ultrasound at 10.5 W/cm 2 /SARAfor 5 to 15 min. It has also been clearly demonstrated in rodents that major fetal brain abnormalities follow systemic maternal hyperthermia of 2.5°C above normal body temperature (Edwards 1986, 1988). Similarly, Hendrickx et al. (1979) showed that raising the core temperature of pregnant monkeys
INTRODUCTION
The use of simple, real-time B-mode ultrasonic imaging in obstetrical examinations is generally considered safe (Wells 1987), given the absence of verified reports of adverse effects on mammalian tissues in more than three decades of clinical use (Ziskin and Pettiti 1988). The low power outputs used in real-time ultrasonic equipment, combined with the brief dwelltime of the beam, provide little opportunity for significant ultrasound-induced temperature increase in tissues during clinical imaging examinations (NCRP 1992; WFUMB 1992). However, currently available equipment with pulsed Doppler capability uses substantially higher power levels (Duck 1990; Duck et al. 1987; Duck and Martin 1991) to allow the detection of signals from small vessels in deep tissue. Furthermore, in a Doppler examination, the ultrasonic beam may be applied to a fixed volume of tissue for extended periods of time. As a consequence, such exposures have the potential to increase significantly the temperature of the tissues being examined. Address correspondence to: Ms. K. L. Bosward, C/-Ultxasonics Laboratory, CSIRO, Division of Radiophysics, 126 Greville Street, Chatswood, NSW, 2067, Australia. 415
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(Macaca radiata) by 2.4°C for 1 h on gestational days 27 to 30 produced a range of fetal malformations. A comprehensive review of the literature has demonstrated that central nervous system tissue is particularly sensitive to temperature elevations (Miller and Ziskin 1989). Due to its higher absorption coefficient for ultrasound, bone is heated during insonation to a greater extent than soft tissue. It is to be expected that tissues adjacent to, or encased in, bone may be significantly heated during pulsed Doppler ultrasonic examinations. Thus, the aim of the present study was to: I. Quantify the extent of temperature increase in the brain of fetal guinea-pigs during exposure to pulsed ultrasound, at intensities and pulsing regimes similar to those available for use during clinical pulsed Doppler examinations. 2. Examine the effect of gestational age and corresponding bone development on the extent and rate of heating of fetal guinea-pig brains during exposure to pulsed ultrasound.
Volume19, Number 5, 1993 The cross-axis intensity profile of the free-field ultrasonic beam in water was plotted in 0.1 mm steps using a 1.0 mm diameter needle-mounted polyvinylidine difluoride (PVDF) hydrophone (NTR Systems Inc., Seattle, USA). Micromanipulators positioned the hydrophone and transducer in a glass tank filled with distilled water and maintained at 38 °C. The profiles were constructed at two specific distances from the transducer (4.4 and 6.0 cm) where the maximum temperature elevation was recorded in tissue for each of the two power levels (260 mW and 1120 mW) used in these experiments (Fig. 1). The spatial peak pulse average (IsPPA) and spatial peak temporal average (IsPTA) intensities were computed from the hydrophone response using a waveform analyser (Data 6100, Data Precision Corp., Danvers, USA). At 6.0 cm distance from the transducer face, the values were 112 W/cm: /SPPAand 2.9 W/cm 2 ISPTA"The - 6 dB beamwidth was 0.27 cm. The average power, measured with a radiometer (Model UPM-DT-l, Ohmic
MATERIALS AND METHODS
~
580
Guinea-pigs (Cavia species) were time-mated to produce fetuses of specific gestational ages. Females were mated immediately following the onset of oestrus, day zero, which was detected by the opening of the vaginal membrane. Twenty-four hours after the onset of oestrus, the female was separated from the male. Pregnancy was diagnosed by the failure to return to oestrus after 16 days and by palpation of the amniotic vesicles at 19 days postoestrus. Pregnant guinea-pigs at 30, 40, 50 and 60 days gestation (dg) were euthanased by an intraperitoneal injection of pentobarbitone sodium (Euthatal 350, May and Baker Australia), and the fetuses were dissected from the uterus. Each fetus was weighed and identified. One fetus from each litter was chosen at random for use as a fresh experimental specimen, while the litter mates were fixed in 10% formalin for at least one week before being used in experiments.
Ultrasonic exposimetry A single lead zirconate titanate transducer with a diameter of 19 mm, radius of curvature of 100 mm, and centre frequency of 3.2 MHz was used in all experiments. The transducer was driven by a pulse function generator (Model 8116A, Hewlett-Packard, USA) and its output varied by means of a regulated power supply and custom-built power amplifier. Pulsing parameters similar to those used in some clinical pulsed Doppler examinations were chosen (pulse duration = 6.25 #s, pulse repetition frequency = 4 kHZ).
W/cm 2
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Fig. 1. Cross-axis intensity profiles of the ultrasonic beam measured at distances of 4.4 and 6.0 cm from the transducer for power levels of 260 mW (upper) and 1120 mW (lower). Solid line curves show the profiles at the positions in the beam where maximum heating was recorded for each power.
Heating of guinea-pig fetal brain @ K. L. BOSWARDel al.
Instruments Inc., MD, USA) at the same axial distance was 260 mW. At the maximum power output used, 1120 mW, the greatest temperature increase was measured at a distance of 4.4 cm. Hydrophone measurements at this location yielded intensities of 91.5 W/cm 2 IsppA and 2.5 W/cm 2 IsvrA and a - 6 dB beamwidth of 0.72 cm.
Experimental procedure Each fetus was secured with surgical tape (Blenderm, 3M, MN, USA) to an acrylic framework over which a sonolucent membrane of surgical film (Vidrape, Parke-Davis, USA) was stretched. The relative positions of the transducer and the fetus were adjusted by micromanipulators and the distance between them was determined by measuring the pulse time delay with a hydrophone and oscilloscope (Model 465B, Tektronix, OR, USA). Temperature was measured using copper-constantan thermocoupies in 26 gauge (0.46 mm) stainless-steel needles (Physitemp Inc, N J, USA) inserted into the fetal tissue and connected to a digital thermometer (BAT 10, Physitemp Inc). Needle mounted thermocouples, rather than bare wires, were chosen to ensure repeatable positioning against the inner aspect of the skull bone. Constant depth of insertion was also reliably achieved. This improved accuracy of positioning was traded against the small risk of error due to viscous heating artifact in measurements made after ultrasound had passed through bone. The absorption of sound in bone is so high as to minimise the effect of localised artifactual heating. Accurate positioning of each thermocouple in the beam was ensured by maximizing the temperature response to an initial lowlevel insonation. The tissue temperature was then allowed to equilibrate to 38°C before recording temperatures at 10 s intervals throughout the 120 s ultrasonic exposure and subsequent cooling periods. In three separate studies, measurements were taken from five fetuses at each gestational age and for each exposure condition. The mean peak temperature levels were calculated from the raw data and recorded in the results. The three studies were as follows.
Single point temperature measurement in fresh and fixed fetal brains. A single thermocouple was inserted to a depth of 0.3 cm in the brain tissue directly under the occipital bone. The ultrasound beam was applied to intercept the brain in the midsagittal plane. The purpose of this experiment was to determine the maximum increase in temperature that occurred in the region of proliferating neural tissue close to bone. Results from fresh and formalin-fixed brains were compared to determine whether the temperature increase was altered significantly by tissue fixation.
417
Multiple temperature measurements in fixed fetal brains. Three thermocouples were inserted, in a transverse plane, through the skull to a depth of 0.3 cm. The tip of the thermocouple closest to the transducer lay in the brain tissue directly under the left parietal bone of the skull. The second thermocouple lay between the cerebral hemispheres, while the third was positioned under the right parietal bone. The aim was to examine further the effect of proximity to bone and the role of the brain in attenuating the ultrasonic beam.
Single point temperature measurement in isolated fixed fetal brains. After removing the brain from the braincase, a single thermocouple was inserted into the midcerebral region. This was done to determine the extent of heating due to absorption by the soft tissue without influence from nearby bone. The mean temperature increases were recorded from six formalin-fixed brains of 60 days gestational age guinea-pig fetuses.
Assessment offetal bone development To obtain a qualitative assessment of the extent of ossification, five fresh fetuses from each gestational age were radiographed in lateral recumbency, using a plastic cassette with a single intensifying screen (KMR, Eastman-Kodak, Rochester, USA) and an autochromatic film (Min R, Eastman-Kodak). The xray equipment (Faxitron 43805N, Hewlett-Packard, Germany) was designed to give high-resolution images of small thicknesses of tissue. In addition, a quantitative measure of the bone mineral content was obtained using an ash determination technique. The fetal skull was removed from the body at the atlanto-occipital joint, homogenized, placed into a preweighed ceramic crucible (Infusil, Bhanu Scientific Instruments Co., Bangalore, India) and dried overnight in an oven at I00-150°C. The crucibles were cooled in a dessicator and the dry-matter weight calculated before being heated to 600°C for 2 h in a muffle furnace (Martin Furnace and Engineering Pty. Ltd., Sydney, Australia). The ash, or total mineral content of the skull, was determined for each fetus and expressed as a percentage of total dry weight.
Statistical analyses All statistical analyses of variance were carried out using the Genstat 4.03e statistical package (1980 Lawes Agricultural Trust, Rothamstaed Experimental Station). The ash determination results were analysed as a completely random design, while all the other studies were analysed as completely random design split-plots. In the studies of heating in fresh versus fixed brains, the whole plots were gestational ages,
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and the different power levels were treated as subplots. In the study of multiple temperature measurements in fixed brains, the whole plots were again the different gestational ages, but the subplots were factorial, including both the different power levels and the different thermocouples. RESULTS
Single point temperature measurement in fetal brains Fresh brains. For each gestational age, a rapid temperature increase was recorded over the initial 40 s, followed by a plateau, and then rapid cooling when the ultrasound exposure ceased. At a power output of 260 mW (2.9 W/cm 2 ISVrA), the position of the beam focus (determined by plotting the - 6 dB beam profile) was 6.7 cm from the transducer, but maximum tissue heating was obtained at a 6.0 cm distance. For a power output of 1120 mW (2.5 W/cm 2 ISVrA), the focal distance was 6.0 cm, but the maximum tissue temperature was recorded at a distance of 4.4 cm. At these distances, the mean peak temperature increases for 260 mW and 1120 mW respectively ranged from 1.3 ° and 4.5°C at 30 dg to 4.7°C and 14.9°C at 60 dg (Table 1). Analysis of variance of the peak brain temperatures revealed significant differences related to the age of gestation (p < 0.01) and ultrasonic power
Table 1. Mean peak temperature increases in fresh and formalin-fixed fetal guinea-pig brains after 120 s exposure to pulsed ultrasound. Mean peak temperature increase
(°C) Gestational age (days) Fresh brains 30 30 40 40 50 50 60 60 Fixed brains 30 30 40 40 50 50 60 60
BPD (cm)
Distance (cm)
260 mW
1120 mW
0.7
4.4 6.0 4.4 6.0 4.4 6.0 4.4 6.0
0.9 1.3 1.1 1.3 2.7 3.5 3.2 4.7
4.5 4.6 4.9 4.2 11.1 10.1 14.9 14.3
1.5 2.0 2.5 0.7 1.5 2.0 2.5
4.4 6.0 4.4 6.0 4.4 6.0 4.4 6.0
4.9 1.4 5.0 1.5 10.8 3.5 13.4 4.2
BPD = biparietal diameter; distance = from transducer to thermocouple in brain under the occipital bone.
Volume 19, Number 5, 1993
level (p < 0.01). The least significant difference (l.s.d.) between the mean peak temperatures for each gestational age, averaged over the two power levels, revealed that the means for the 30 and 40 dg fetuses were significantly lower (p < 0.05) than those for the 50 and 60 dg fetuses. Whereas the means for the 30 and 40 dg fetuses were not significantly different from each other, the difference between the means of the 50 and 60 dg fetuses was significant (p < 0.05).
Fixed brains. The mean peak temperature for 260 mW and 1120 mW exposures ranged from 1.4 ° and 4.9°C, for fetuses of 30 dg, to 4.2 ° and 14.1 °C for those of 60 dg (Table 1). As in the fresh brains, a rapid rise in temperature was followed by a plateau, and rapid cooling when insonation ceased (Fig. 2). The shape of the plateau phase of the heating curve is illustrated in Fig. 3 which shows results of 3 min exposures to 1120 mW power. There is minimal increase in the mean temperature levels beyond the 2 min exposure duration. For ease of correlation, all data shown in the tables of results represent mean values obtained after exposure durations of 2 min. Analysis of variance of the peak temperatures revealed significant differences related to gestational age (p < 0.01) and ultrasonic power level (p < 0.01). The 1.s.d. between the means for each gestational age, averaged over the two power levels, revealed that the means for the 30 and 40 dg fetuses were significantly lower than those for the 50 and 60 dg fetuses (p < 0.05). Analysis of variance of the peak temperature in fixed and fresh fetal brains revealed no significant difference between the means at either power level or gestational age. Multiple temperature measurements in fixed fetal brains The highest temperature was consistently recorded from the thermocouple located in the brain tissue under the parietal bone closest to the transducer (Table 2, thermocouple 1). The mean peak temperature increases after 120 s exposure to 260 mW ranged from 1.2°C at 30 days to 5.2°C at 60 days gestational (dg). The respective values for exposure to 1120 mW were 3.4 ° and 15.9°C. The largest temperature increases were always observed in 60 dg fetuses. Measurements from the thermocouples in the midcerebral region yielded temperature increases at the same gestational ages ranging from 0.6 ° to 0.8°C at 260 mW, and 2.5 ° to 3.4°C at 1120 mW power. In this situation, the greatest temperature increase was measured in 50 dg fetuses (Table 2), where the mean peak temperatures for 260 mW and 1120 mW power outputs were 0.9 ° and 4.4°C, respectively.
Heating of guinea-pig fetal brain • K. L. BOSWARDel
419
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Fig. 2. Mean temperature elevations in fixed guinea-pig fetal brain tissue, adjacent to the occipital bone, during 120 s exposures to 260 mW pulsed ultrasound. Distance = 6.0 cm from the transducer.
Similarly, results from the thermocouple placed under the distal parietal bone o f the skull showed the largest temperature increases to occur in 50 dg fetuses, ranging from 1.5 ° to 5.6°C for 260 m W and 1120 m W , respectively (Table 2). T h e smallest t e m p e r a t u r e increase (0.9°C) was recorded in the distal thermocouple in 60 dg fetuses exposed to 260 m W power output. T e m p e r a t u r e s measured from thermocouples close to the proximal (Fig. 4) and distal parietal bones
rose rapidly at both power levels over the initial 40 s, followed by a plateau, and rapid cooling when the ultrasound exposure ceased. The temperature in the midbrain increased at a slower rate without reaching the same plateau region, and cooling was slower. Analysis o f variance revealed a significant difference between the m e a n peak temperatures at different power levels for all gestational ages (p < 0.05 at 30 and 40 d g ; p < 0.01 at 50 and 60 dg). The l.s.d, o f the m e a n peak temperatures showed that at 30, 50 a n d 60
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Ultrasound in Medicine and Biology
Volume 19, Number 5, 1993
Table 2. M e a n peak temperature increases in fixed fetal guinea-pig brains after 120 s exposure to pulsed ultrasound in the transverse plane. Mean peak temperature increase (°C) Gestational age (days)
Bone thickness (mm)
Axial distance (cm)
th. 1
th. 2
th. 3
0.17 0.23 0.30
6.0 6.0 6.0 6.0
1.2 1.6 3.7 5.2
0.6 0.9 0.9 0.8
1.0 1.1 1.5 0.9
Power = 260 mW 30 40 50 60 Isolated brain 60 Power = 1120 mW 30 40 50 60 Isolated brain 60
6.0 0.17 0.23 0.30
4.4 4.4 4.4 4.4
0.00
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2.6 3.4 4.7 11.7 15.9
2.5 3.9 4.4 3.4
3.1 4.3 5.6 3.6
7.7
th. 1 = thermocouple positioned under the proximal parietal bone; th. 2 = middle of cerebral hemispheres; th. 3 = under the distal parietal bone; distance = transducer to thermocouple.
Assessment offetal bone development
dg, the increase at the first thermocouple was significantly different (p < 0.05) from that at the second and third thermocouples.
Fetal radiography. Radiographs of the 30 dg fetuses showed the beginnings of ossification in the ribs, maxilla, mandible and the orbital area. At 40 dg, ossification had progressed to include the vertebrae, with further development of the radius, humerus, scapula, femur and tibia. The skull showed thickening of the bones of the braincase, orbit, mandible and maxilla. The tympanic bullae were prominent. By 50 dg, the appendicular skeleton had developed significantly
Insonation of isolatedfixed fetal brain A mean peak temperature increase of 2.6°C occurred in brains of 60 dg fetuses after insonation for 120 s (Fig. 5) at 260 mW power when positioned 6.0 cm from the transducer. When the power output was increased to 1120 mW, the mean peak temperature increase was 7.7°C (Table 2). 6¸ 5.
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Fig. 4. Mean temperature elevations in fixed guinea-pig fetal brain tissue during 120 s exposures to 260 m W pulsed ultrasound. A single thermocouple was placed under the parietal bone at a distance o f 6.0 cm from the transducer.
Heating of guinea-pig fetal brain • K. L. BOSWARDet al.
421
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increase (°C)
0 0
60
~ 120
180 240 Time (secs)
300
360
Fig. 5. Mean temperature increases in the midcerebral region of 60 dg fetal guinea-pig brains isolated from the cranium and exposed to pulsed ultrasound at 260 mW and 1120 mW power.
and each vertebra was prominent. The teeth in both the mandible and maxilla were evident and the tympanic buUae were especially thickened. At 60 dg the skeleton was almost completely ossified. Fetal ash determination. The ash content of the skull increased significantly (p < 0.01) with gestational age (Fig. 6). The 1.s.d. showed that the ash content at each gestational age was significantly different from all the other ages (p < 0.05). DISCUSSION
ture change increased significantly with advancing gestational age. It is uncertain how much of the enhanced temperature increase was due to a larger area of bone absorbing more ultrasound. In the present study, the results of fetal ash assay demonstrated progressive increase in mineralisation consistent with increasing gestational age from day 30 onwards; it is known that ossification in the guineapig fetus begins on the 27-28th day (Graham and Scothorne 1970). The large difference in the maximum brain heating that occurred between 40 and 50 dg is apparently due to significant bone development
This study has demonstrated significant t e m p e r a ture increases in brain tissue adjacent to bone. Mean temperature increases of 4.7°C (occipital bone) and 5.2°C (parietal bone) were recorded in 60 dg fetuses, after 120 s exposure to an intensity of 2.9 W/cm 2 IsrrA (260 mW). A recent study reported a temperature increase of 5.6°C in the cranial bone in young mice exposed for 45 s to focussed pulsed ultrasound with an ISPTAof 1.5 W/cm 2 (Carstensen et al. 1990). Temperature measurements were made at the front surface of the 0.5 mm thick mouse skull bone, while our results were obtained at the bone/soft tissue interface inside the 0.3 mm thick guinea-pig skull bone. It is possible that our results may have been underestimated due to heat conduction along the thermocouple needle. In an in vitro study of heating in human fetal femurs (Drewniak et al. 1989), a temperature rise of 4.0°C was recorded in the diaphysis of a 108 dg femur after 60 s exposure to CW ultrasound at an IsrrA of 1.0 W / c m 2. In this latter study, the magnitude of tempera-
% ash
30
40
50
60
Gestational age (days) Fig. 6. Total ash content of guinea-pig fetal skulls expressed
as a percentage of dry skull weight, and plotted as a function of advancing gestational age.
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providing a larger volume of bone (increased skull diameter and thickness) for ultrasound absorption in the older fetuses. Fetal radiography provided qualitative confirmation of the progression in bone development and supported the data obtained from ash determination. During ultrasonic exposure, the pattern of brain heating was similar for insonations in the sagittal and transverse planes, with the highest temperatures generally being recorded in the older fetuses. The peak temperature elevations in the transverse approach resulted primarily from absorption of ultrasound by the parietal bones, with conduction of heat to the adjacent soft tissue. At each gestational age, the temperature increase in the middle of the cerebral hemispheres was lower than that recorded near bone. The slower rate of temperature rise suggests conduction of heat from the parietal bones in addition to local heating from the absorption of ultrasound by the brain tissue. The resultant heating of the central brain depends on the extent of bone formation and ultrasound attenuation. In late gestation (60 dg), thicker bone attenuates the incident ultrasound beam. This reduces the energy level available for absorption by soft tissue, while rapidly generating heat that can be conducted into the brain tissue. The effect of bone thickness on heating in equivalent volumes of brain tissue is demonstrated by the factor of three increases in soft tissue heating when the brain was insonated after removal from the cranium of 60 dg fetuses. The mean peak temperature in the brain tissue close to bone was found to increase with advancing gestational age and increased ultrasound exposure. The temperature increase was also a function of the distance of the specimen from the transducer, where maximum tissue heating occurred at an axial position closer to the transducer than the geometric focus of the beam. For a power output of 260 mW, the position of the beam focus was 6.7 cm from the transducer, but maximum tissue heating was obtained at a distance of 6.0 cm; for a power output of 1120 mW the focal distance was 6.0 cm, but the maximum tissue temperature was recorded at 4.4 cm. This finding is similar to a report of greater heating "in front of the focus" in soft tissue-mimicking gels (Bacon and Carstensen 1990) under conditions of nonlinear propagation. Furthermore, the present findings have questioned the relevance OflsrrA intensity as a predictor of tissue heating. In this study, the measured value of the IsP'rA (and ISPPA)was similar for power outputs of 260 mW and 1120 mW due to differences in beamwidth with distance from the transducer. However, heating was greater at 1120 mW where there was a wider
Volume19, Number 5, 1993 heated area. For the narrower beam at 260 mW and 6 cm distance, a greater thermal gradient would allow more rapid heat dissipation from the axis. The situation is further complicated by nonlinear processes which increase as a function of distance and power and provide opportunities for shock-loss to the medium. In in vitro studies, ter Haar et al. (1989) also reported that neither IsrrA nor /SARA showed good correlation with the temperature increase during a 1 min insonation of excised liver. The intensity values measured under free-field conditions in water are not the same as the in situ values that are subjected to attenuation from the tissue. In addition, the nonlinear distortion associated with higher peak positive pressure amplitudes leads to enhancement of the predicted temperature rise due to the absorption of the higher harmonic components (Swindell 1985). In the present study, the width of the ultrasound beam played a role in determining the temperature obtained in the tissue. The beam width measured at 6.0 cm axial distance for a power of 260 mW was 0.27 cm, while at 4.4 cm and 1120 mW it was 0.72 cm. The wider beam at 1120 mW means that the IsrrA is low but a larger area of tissue is heated. The effects of nonlinearity, with higher peak positive pressure amplitudes and the associated absorption of higher harmonic components, may also be responsible for the greater differences in the temperature increases recorded at the higher exposure levels. The shape of the graph of temperature increase versus time is asymptotic. Initially, the slope is steep with more than 75% of the temperature rise occurring in the initial 40 s when temperature increases linearly with time in proportion to the absorbed energy. This is followed by a slower rate of temperature rise as heat is conducted from the exposed area of tissue. In the final phase the curve flattens, representing the establishment of a thermal equilibrium as heat is dissipated. The effectiveness of the water tank (maintained at a constant temperature of 38°C) in dissipating heat is shown by the rapid decrease in brain temperature that occurs when the ultrasonic exposure ceased. This cooling results from thermal conduction, as a temperature gradient is established between the heated tissue and the water tank. Thermal conduction occurs as soon as a temperature gradient is established in a tissue, and the heat diffuses away in an attempt to eliminate the gradient. In situ cooling comes from two main sources: thermal conduction and tissue perfusion. Although equations for a cooling function have been proposed (WFUMB 1992), it is difficult to estimate the effect of perfusion on heat dissipation, since vascularity varies between and within tissues at any one time, depending on the organ and physiologi-
Heating of guinea-pig fetal brain • K. L. BOSWARDel al.
cal state. In vivo studies on the cooling effect of blood circulation in fetal tissues exposed to ultrasound have reported smaller temperature increases in perfused tissue than in avascular tissue (Abraham et al. 1989; Sikov et al. 1984). The effect of tissue perfusion and its role in heat dissipation, compared to the relatively large volume of temperature-stabilised water in a tank, requires further investigation. Few studies have determined the temperature rise in fetal tissue during exposure to ultrasound. Abraham et al. (1989) measured temperature increases in rat fetuses at 15-20 dg, and reported an increase of 10.0°C in fixed fetuses after exposures of l0 min to CW/SARA intensity of 4.2 W / c m 2. In live fetuses the same exposure gave temperature elevations of I 1.0 ° and 12.8°C with and without blood circulation, respectively. Sikov et al. (1984) measured the temperature in the exteriorised uterine horns of 9 dg rats. During 15 min exposures to CW ultrasound, at intensities of 1.8 to 20 W / c m 2, it was shown that ligation of ovarian and uterine arteries resulted in a greater temperature rise. During in vivo exposures of mouse embryos, prior to bone development, Fry (1986) observed greater heating from pulsed-wave (PW) than from CW ultrasound. Exposure for 20 s to an intensity of 27.0 W / c m 2 ISPTA gave a maximum temperature rise of 4. l °C with CW, and 8.0°C with PW ultrasound. The result was attributed to the highly nonlinear sound field conditions associated with the pulsing regime. The significant heating of fetal brain found in the present study occurred with ultrasonic pulsing conditions, power outputs and ISPTAvalues similar to the range of values reported for modem pulsed Doppler equipment (Duck et al. 1987; Duck 1989, 1990; Stewart et al. 1986). Although a higher average power output (l 120 mW) was used for the upper level, its average intensity (IsrrA) value, expressed over the larger beamwidth, was similar to that for 260 mW in the narrower beam. In the present in vitro study, the fetuses were dissected free from the uterus. In vivo, the matemai abdominal wall would attenuate ultrasonic energy and smaller elevations in the temperature of the central nervous system tissues might be expected. If, however, an acoustically transparent window, such as the bladder, were used during a pulsed Doppler ultrasonic obstetric examination, minimal loss of power would occur once the ultrasonic beam had passed through the maternal abdominal wall. Rather than attenuating, these fluids offer an opportunity for the development of a shock front that may enhance heating effects of pulsed ultrasound beams (Bacon 1984). Thus, there is a need for further in vivo observations to determine what, if any, effect the attenua-
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tion of the ultrasonic beam by the maternal abdominal wail, or other overlying structures, has on temperature elevations in fetal tissues. The central nervous system is known to be especially sensitive to heat (Edwards 1986) and temperature elevations of 2 ° to 2.5°C are teratogenic to the developing nervous tissue in experimental animals (Edwards 1988). The nature of the biological effect depends on the duration of the hyperthermic exposure and the stage of pregnancy when it occurred. Reported defects include talipes, arthrogryposis, microphthalmia, micrencephaly, neural tube defects and other functional defects (Edwards 1988). Using an in vitro whole-embryo culture system, Barnett et al. (1990) exposed rat embryos at 9.5 dg to pulsed ultrasound and elevated temperature. Effects included decreased somite number, significant reductions in the ratios of brain:body area, and a characteristic oedematous-blistering of the prosencephalon, after exposure to ISPTA 1.2 W / c m 2 together with a 1.5°C temperature elevation for 15 min. A review of the literature on mammalian hyperthermia (Miller and Ziskin 1989) showed that the higher the temperature, or the longer the hyperthermic exposure, the greater the chance of damage occurring in mammalian tissue. Accordingly, an absolute temperature of 43.2°C, as observed in the present study following 2 min exposure to 2.9 W / c m 2/SPTA, would need to be maintained for 1 min for significant levels of damage to occur. Similarly, l0 min at 41.5°C would also be expected to cause tissue damage. The present findings were obtained under a specific set of experimental conditions which did not take account of attenuation by the maternal abdominal wall and cooling by vascular perfusion. It is unlikely that these conditions would be duplicated during routine pulsed Doppler clinical examinations. Ultrasonologists and ultrasonographers should be aware, however, of the possible damaging effects of hyperthermia. Care should be taken to minimize the dwell-time of the ultrasonic beam on the fetal tissue and to minimize the power outputs used with pulsed Doppler equipment in clinical examinations. Acknowledgement--The financial support of the Selby Scientific Foundation is gratefully acknowledged.
REFERENCES Abraham, V.; Ziskin, M. C.; Heyner, S. Temperature elevation in the rat fetus due to ultrasound exposure. Ultrasound Med. Biol. 15:443--449; 1989. Bacon, D. R. Finite amplitude distortion of the pulsed fieldsused in diagnostic ultrasound. Ultrasound Med. Biol. 10:189-195; 1984. Bacon, D. R.; Carstensen E. L. Increased heating by diagnostic ul-
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trasound due to nonlinear propagation. J. Acoust. Soc. Am. 88:26-34; 1990. Barnett, S. B.; Walsh, D. A.; Angles, J. A. Novel approach to evaluate the interaction of pulsed ultrasound with embryonic development. Ultrasonics 28:166-170; 1990. Carstensen, E. L.; Child, S. Z.; Norton, S.; Nyborg, W. L. Ultrasonic heating of the skull. J. Acoust. Soc. Am. 87:1310-1317; 1990. Carstensen, E. L.; Gates, A. H. Ultrasound and the fetus. In: Nyborg, W. L.; Ziskin, M. C., eds. Biological effects of ultrasound. Clinics in diagnostic ultrasound, Vol. 16. New York: Churchill Livingstone; 1985:85-95. Child, S. Z.; Carstensen, E. L.; Davis, H. A test for the effects of low temporal average intensity pulsed ultrasound on the rat fetus. Exp. Cell Biol. 52:207-210; 1984. Child, S. Z.; Carstensen, E. L.; Gates, A. H.; Hall, W. J. Testing for the teratogenicity of pulsed ultrasound in mice. Ultrasound Med. Biol. 14:493-498; 1988. Drewniak, J. L.; Carues, K. I.; Dunn, F. In vitro ultrasonic heating of fetal bone. J. Acoust. Soc. Am. 86:1254-1258; 1989. Duck, F. A. Output data from European studies. Ultrasound Med. Biol. 15 (Suppl. 1):61-64; 1989. Duck, F. A. Two case studies in ultrasound exposure measurements. Br. J. Radiol. 63:392; 1990. Duck, F. A.; Martin, K. Trends in diagnostic ultrasound exposures. Phys. Med. Biol. 15:1423-1432; 1991. Duck, F. A.; Starritt, H. C.; Anderson, S. P. A survey of the acoustic output of ultrasonic Doppler equipment. Clin. Phys. Physiol. Meas. 8:39-49; 1987. Edwards, M. J. Hyperthermia as a teratogen: A review of experimental studies and their clinical significance. Teratogenesis Carcinog. Mutagen. 6:563-582; 1986. Edwards, M. J. Hyperthermia and the developing central nervous system. Congen. Anomal. 28 (Suppl.):S119-S133; 1988. Fry, F. J. Effects of ultrasound exposure on pregnancy: A study in the mouse. IEEE Trans. Ultrason. Ferroelectr. Freq. Contr. 33:225-234; 1986. Graham, R. W.; Scothorne, R. J. Calcium homeostasis in the fetal guinea-pig. Q. J. Exp. Physiol. 55:44-53; 1970. Hendrickx, A. G.; Stone, G. W.; Henrickson, R. V.; Matayoshi, K.
Volume 19, Number 5, 1993 Teratogenic effects of hyperthermia in the bonnet monkey (Macaca radiata). Teratology 19:177-182; 1979. Miller, M. W.; Ziskin, M. C. Biological consequences ofhyperthermia. Ultrasound Med. Biol. 15:707-722; 1989. NCRP. Exposure criteria for medical diagnostic ultrasound: 1. Criteria based on thermal mechanisms. Report No. 113. Bethesda, MD: National Council for Radiation Protection and Measurements; 1992. Shoji, R.; Murakami, U.; Shimizu, T. Influence of low-intensity ultrasonic irradiation on prenatal development of two inbred mouse strains. Teratology 12:227; 1975. Sikov, M. R.; Collins, D. H.; Cart, D. B. Measurement of temperature rise in prenatal rats during exposure of the exteriorized uterus to ultrasound. IEEE Trans. Sonics Ultrason. SU-31:497503; 1984. Sikov, M. R.; Hildebrand, B. P. Effects of ultrasound on the prenatal development of the rat. Part 1.3.2 MHz continuous-wave at nine days of gestation. J. Clin. Ultrasound 4:357-363; 1976. Stewart, H. F.; Silvis, P. X.; Smith, S. W. Patient exposure data for Doppler studies. Clin. Diagn. Ultrasound 17:187-195; 1986. Swindell, W. A theoretical study of nonlinear effects with focused ultrasound in tissues: An acoustic bragg peak. Ultrasound Med. Biol. 11:121-30; 1985. Takabayashi, T.; Sato, S.; Sato, A.; Ozawa, N.; Sou, S.; Yajima, A.; Suzuki, M. Influence of pulse-wave ultrasonic irradiation on the prenatal development of mouse. Tohuku J. Exp. Med. 147:403-410; 1985. ter Haar, G. R.; Duck, F.; Daniels, F. A.; Starritt, H. Biophysical characterisation of diagnostic ultrasound equipment--Preliminary results. Phys. Med. Biol. 34:1533-1542; 1989. Wells, P. N. T. The safety of diagnostic ultrasound. Br. J. Radiol. (Suppl. 20). London: British Institute of Radiology; 1987. WFUMB. World Federation for Ultrasound in Medicine and Biology Symposium on Safety and Standardisation in Medical Ultrasound: Issues and recommendations regarding thermal mechanisms for biological effects of ultrasound. Barnett, S. B.; Kossoft, G., eds. Ultrasound Med. Biol. 18:(9); 1992. Ziskin, M. C.; Pettiti, D. B. Epidemiology of human exposure to ultrasound: A critical review. Ultrasound Med. Biol. 14:91-96; 1988.