Br. J. Cancer (1982) 45, Suppl. V, 151

ULTRASONICALLY INDUCED CAVITATION IN VIVO

G. TER HAAR,* S. DANIELS,t K. C. EASTAUGHt AND C. R. HILL*From the *Department of Physics, Institute of Cancer Research, Royal Marsden Hospital,

Sutton, Surrey and the tDepartment of Pharmacology, South Parks Road, Oxford

IT HAS been established that cavitationis an important mechanism for producingeffects in biological systems irradiatedwith ultrasound in vitro (Hill et al., 1969;Morton et al., 1982). However, evidencefor cavitation activity in vivo as a resultof ultrasound irradiation is sparse. In thispaper, cavitation is defined as the forma-tion, growth and activity of gas-filledcavities. Evidence is presented for theappearance of such cavities in vivo in thehind limb of anaesthetized guinea-pigsduring irradiation with 0 75 MHz ultra-sound. An 8 MHz ultrasonic imagingsystem (Daniels et al., 1979) has been usedto detect these gas cavities and to studytheir distributions in space and time(Daniels et al., 1980). The imaging systemis capable of detecting gas bubbles withdiameters > 10 ,im, although no accuratemeasurements of bubble diameter can bemade (Beck et al., 1978).

METHODS

Animal preparation.-Male guinea-pigs(400-600 g bodyweight) were used for theseexperiments. Before each experiment theguinea-pigs were anaesthetized with urethane(1.5 g/kg-1 bodyweight, i.p.), shaved roundthe hindquarters, chest and neck, and allresidual hair from the left hind limb removedusing a proprietory depilatory cream. Electro-cardiogram (e.c.g.) electrodes were attached,one positioned directly above the heart, theother at the back of the neck. Finally, theguinea-pigs were positioned in a Perspexholder with the left hind limb extendeddownwards away from the body and theright hind limb lying along the line of thebody. Both limbs were securely taped inposition, care being taken not to obstructblood flow.Imaging system.-High resolution, bistable

B-scans were obtained using a mechanicallyswept, focused 5 mm diameter probe. Theprobe assembly was supported above arectangular tank such that it could bepositioned accurately in three dimensions.The Perspex holder containing the guinea-pig was fixed at the front of the tank and theultrasound probe located centrally and 15 mmbehind the extended left hind limb. Acousticcoupling was achieved with a salt solution(0.7% NaCl; 0 035% KCI; 0.03% MgSO4.7H20: W/V). The gain, suppression andswept gain of the system were adjusted togive an image with a minimum of fine tissuedetail. This facilitated subsequent imageanalysis. Alternate images were automatic-ally recorded, at a rate of one every 2 sec, on35 mm film (Kodak 2495 RAR). The entirescanning assembly, shown in Fig. 1, fittedinside a 36 litre pressure chamber.

0 75 MHz ultrasound irradiation.-A RankMultiphon Mark II ultrasonic generator,fitted with a 2-5 cm diameter transducer,was used to irradiate the left hind leg of theguinea-pig with 0 * 75 MHz ultrasound. Theintensity was continuously variable up to3 Wcm-2 (spatial average). The transducerwas positioned in the scanning tank per-pendicular to the imaging probe (in the samehorizontal plane) and 4 cm from the leg. Thetransducer was located vertically so that theimaged tissue plane fell in the centre of thetherapy beam (Fig. 1). Control experimentsestablished that no interaction between thetwo beams could be observ-ed for 0 75 MHzultrasound intensities < 2 Wcm-2. The beamprofile for the 0'75 MHz ultrasound is shownin Fig. 1.Image analysis.-Each film recorded from

an experiment consisted of a 5 min (150recorded images) control sequence followedby images recorded during 0-75 MHz ultra-sound irradiation, with or without the applica-tion of increased ambient pressure. Analysisconsisted of: (i) combination of all the imagesobtained during the control period, to produce

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(DT) and 0-75 MHz irradiation probe (TT). Coil, shown at the bottom of the tank, thermostaticallycontrols the temperature of the acoustic coupling medium to 370C. (B) Beam profile of 0O75 MHzultrasound. (C) Bistable ultrasound image recorded from guinea-pig leg; SK, skin surface; TB,tibula; FB, fibula; remaining internal echoes result from intermuscular interfaces. Direction oftransducers indicated, DT, 8 MHz imaging probe; TT, 075 MHz irradiation probe. Time isindicated at the bottom of the image, displayed in min sec.

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ULTRASONICALLY INDUCED CAVITATION

a "master control" which takes account ofvariations in the images due to slight move-ments of the leg; (ii) comparison of all suc-cessive images, in turn, with the mastercontrol, to identify all new echoes, and(iii) plotting of the location of the sites ofappearance of new echoes, relative to thelocation of pre-existing tissue interfaces,together with the time and duration of theirappearance. For those experiments in whichthe ambient pressure was raised to 5-5 bargauge (0-55 MPa) during 0-75 MHz ultra-sound irradiation a further 30 images,recorded from the start of compression, wereincluded in the master control to account foradditional movements of the leg caused bypressurization.

RESULTS

It has previously been shown (ter Haar& Daniels, 1981) that the number of sitesof appearance and the number of newechoes is proportional to the intensity andtime of irradiation with 0-75 MHz ultra-sound. The experiments described in thispaper were designed to establish whetherthese new echoes are the result of cavita-tion. Raising the ambient pressure aftera period of ultrasound irradiation will, ifnew echoes are due to gas bubbles, causethese echoes to disappear. Thus, theseexperiments consisted of image recordingduring: (a) 5 min control period, (b) 5 minirradiation with 0-75 MHz ultrasound at680 m Wcm-2 (spatial average), (c) com-pression to and maintenance at either 4or 5-5 bar gauge (0.4 or 0-55 MPa) for5 min with continued 0-75 MHz ultra-sound irradiation, (d) maintenance atpressure without 0-75MHz ultrasoundfor 5 min and (e) decompression to, andmaintenance at, atmospheric pressure for

i.5mm.Initially, 3 experiments involving pres-

surization to 4 bar gauge were performed.However, due to incomplete recording ofthe images a full temporal analysis ofthese experiments was not possible. Spatialanalysis was completed and is includedin Fig. 2. The full results of the 3 experi-ments involving compression to 5-5 bargauge are shown in Figs 2 and 3. An event

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Time (min)FIG. 2.-Rate of accumulation of sites of

bubble formation. 0-5 min, 0-75 MHz ultra-sound, 680 mWcm-2 (7 animals); 5-10 min,0-75 MHz ultrasound, 680 mWcm-2, 5-5bar gauge (6 animals); 10-15 min, Ultra-sound off, 5-5 bar gauge (6 animals); 15-20min, Ultrasound off, atmospheric pressure(5 animals).

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FIG. 3.-Histograms showing the total num-ber of events in each 60 sec interval for oneanimal. Shaded areas of the histogramrepresent events that are "persistent" (seetext). See legend of Fig. 2 for experimentaltiming.

is defined as any record of a new echo ata single site. The duration of eventsranges from those present on only a singleimage (single frame events) to thoserecorded on several successive images(persistent events). Events are equatedwith individual gas bubbles (Daniels et al.,1980). Single frame events are believed torepresent intravascular bubbles passingthrough the plane of scan. Persistentevents may represent either intra- orextravascular gas bubbles.The spatial analysis (Fig. 2) shows that

the recruitment of sites at which eventsare seen is rapid, > 50% of the final

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G. TER HAAR, S. DANIELS, K. C. EASTAUGH AND C. R. HILL

number of sites being seen within thefirst minute. The total number of sitesrecruited during 5 min irradiation with0 75MHz ultrasound at 680 mWcm-2ranges from 10 to 65 (mean 36-7, standarddeviation 22.3). This variation may reflectthe variation in leg size (related to body-weight) and in the exact vertical locationof the imaging probe. Recruitment of sitesceased with the application of pressure.The temporal analysis (Fig. 3) shows

that the total number of events, recordedduring 1 min periods of irradiation, wasroughly constant over the 5 min irradia-tion period. The total number of eventsrecorded during the first minute waswithin 15% of the mean total for thewhole period. The application of pressurecaused an immediate reduction in thenumber of events (mean total duringirradiation 41-5 events; mean total duringirradiation plus pressure 6.8). Finally, achange in the type of events recorded wasobserved. During irradiation with 0 75MHz ultrasound approximately 50% ofall events were single frame events, bothwith and without pressure. However, inthe absence of irradiation with 0 75 MHzultrasound the proportion of single frameevents fell to 15%, only beginning to riseafter decompression. This may suggestthat the most susceptible area for cavita-tion is intravascular and that even atpressure a small number of intravascularbubbles can be generated. The residual"events" during the period at pressurewithout 0*75 MHz ultrasound irradiationmay represent the "noise" level. Alter-natively, it is also possible that even theseevents, which persist after pressurizationand in the absence of 0 75 MHz ultra-sound irradiation, still represent stationarygas bubbles stabilized by some structuralmechanism (e.g. Philp et al., 1971;Harvey, 1951).

DISCUSSION

These results show that a considerablenumber of new echoes arise during irradia-tion with 0 75 MHz ultrasound at 680

mWcm-2 and that a majority of thesenew echoes are caused to disappear byincreasing the ambient pressure. Thissusceptibility to pressure strongly sug-gests that these echoes are due to gasbubbles within the leg. Furthermore, thetemporal distribution of these echoes,leading to the differentiation of singleframe and persistent events, suggests amixture of intravascular and extravascularbubbles.The rapidity of onset of activity, on the

time scale of observation, is consistentwith the idea that the 0 75 MHz ultra-sound is the causal agent. It is perhapssurprising that in some experiments (Fig.2) evidence is seen for a continuation ofrecruitment of sites throughout the 5 minperiod of irradiation. This suggests anumber of different sites for cavitation,each having a different activation energy.If this is so, it is not known whether thesame initiation mechanism operates ateach site.The bubbles observed may constitute a

biological hazard either directly or in-directly. Extravascular bubbles may causetissue disruption and damage, and station-ary intravascular bubbles may cause localischaemia and activation of blood clottingmechanisms. There have been some indica-tions in the early literature (e.g. Grabar,1953) that ultrasonic irradiation oftumours may sometimes promote meta-stasis and it seems possible that in vivocavitation could have the effect of dis-persing malignant cells into blood.

Restricted, as in these experiments, toa peripheral region these effects may notprove to represent any significant hazard.However, mobile intravascular bubblesare potentially a more serious hazard.Arriving centrally they may disturbcardiac or respiratory function or, ifintroduced into the blood supply ofnervous tissue, they may cause ischaemicdamage and consequent neurological symp-toms. From these experiments it is pos-sible to estimate the volume of free gasrepresented by the observed single frameevents. The total mean number of single

154

ULTRASONICALLY INDUCED CAVITATION 155

frame events over the 5 min irradiationperiod was 108. They all fell into acategory, defined elsewhere (Daniels et al.,1980), that represents bubbles with dia-meters between 10 and 100 yim. Assumingan average diameter of 50 ,um the freevolume of gas would be 7 x 10-3 d or,if the maximum radius was assumed,5-7 x 10-2 ,l. Even if the true number ofsingle frame events were 2 orders ofmagnitude higher, from a considerationof the intermittent nature of the observa-tions (Daniels et al., 1980), the freevolumes would still be 0 7 pl or 5-7 plrespectively. This correction factor isunrealistically high, but even so as anupper limit, suggests that the volume offree gas involved is very small.

In conclusion we have demonstratedthat free gas bubbles are induced withinliving mammalian tissue by 0 75 MHzultrasound irradiation at 680 mWcm-2.For 5 min irradiations we have observedno specific indications ofassociated hazard,but some possibily hazardous outcomes ofthe phenomenon can be envisaged andmay justify further investigation.

REFERENCES

BECK, T. W., DANIELS, D., PATON, W. D. M. &SMITH, E. B. (1978) The detection of bubbles indecompression sickness. Nature, 276, 173.

DANIELS, S., PATON, W. D. M. & SMITH, E. B.(1979) An ultrasonic imaging system for the studyof decompression induced gas bubbles. UnderseaBiomed. Res., 6, 197.

DANIELS, D., DAVIES, J. M., PATON, W. D. M. &SMITH, E. B. (1980) The detection of gas bubbl4in guinea-pigs after decompression from airsaturation dives using ultrasonic imaging. J.Physiol., 308, 369.

GRABAR, P. (1953) The biological action of ultra-sonic waves. Adv. Bio. Med. Physics, 3, 191.

HARVEY, E. N. (1951). Physical factors in bubbleformation. In Decompression Sickness. Ed. J. F.Falton. Philadelphia: W. B. Saunders Co. p. 90.

HILL, C. R., CLARKE, P. R., CROWE, M. R. &HAMMICK, J. W. (1969) Biophysical effects ofcavitation in a 1 MHz ultrasonic beam. Proc.Conf. Ultrasonics for Industry. London: Illiffe.p. 26.

MORTON, K., TER HAAR, G., STRATFORD, I. J. &HILL, C. R. (1982) The role of cavitation in theinteraction of ultrasound with V79 Chinesehamster cells in vitro. Br. J. Cancer, 45, Suppl.V,147.

PHILP, B. R., SCHACHAM, P. & GOWDEY, C. W.(1971) Involvement of platelets and micro-thrombi in experimental decompression sickness:similarities with disseminated intravascular coagu-lation. Aerospace Med., 42, 494.

TER HAAR, G. & DANIELS, S. (1981) Evidence forultrasonically induced cavitation in vivo. Phys.Med. and Biol., 26, No. 6 (Nov.).