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An experimental system for the study of ultrasound exposure of isolated blood vessels
This article has been downloaded from IOPscience. Please scroll down to see the full text article.
2013 Phys. Med. Biol. 58 2281
(http://iopscience.iop.org/0031-9155/58/7/2281)
Download details:
IP Address: 193.1.100.108
The article was downloaded on 22/04/2013 at 08:17
Please note that terms and conditions apply.
View the table of contents for this issue, or go to the journal homepage for more
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An experimental system for the study of ultrasoundexposure of isolated blood vessels
Anna Tokarczyk1, Ian Rivens1, E van Bavel2, Richard Symonds-Tayler1
and Gail ter Haar1
1 Therapeutic Ultrasound, Joint Department of Physics, Royal Marsden NHS Foundation Trust:Institute of Cancer Research, Downs Road, Sutton, Surrey, SM2 5PT, UK2 Department of Biomedical Engineering and Physics, Academic Medical Center, PO Box 22660,1100 DD Amsterdam, The Netherlands
Received 29 July 2012, in final form 9 February 2013Published 12 March 2013Online at stacks.iop.org/PMB/58/2281
AbstractAn experimental system designed for the study of the effects of diagnosticor therapeutic ultrasound exposure on isolated blood vessels in the presenceor absence of intraluminal contrast agent is described. The system comprisedseveral components. A microscope was used to monitor vessel size (and thusvessel functionality), and potential leakage of intraluminal 70 kDa FITC-dextran fluorescence marker. A vessel chamber allowed the mounting of anisolated vessel whilst maintaining its viability, with pressure regulation for thecontrol of intraluminal pressure and induction of flow for the infusion of contrastmicrobubbles. A fibre-optic hydrophone sensor mounted on the vessel chamberusing a micromanipulator allowed pre-exposure targeting of the vessel to within150 μm, and monitoring of acoustic cavitation emissions during exposures.Acoustic cavitation was also detected using changes in the ultrasound drivevoltage and by detection of audible emissions using a submerged microphone.The suitability of this system for studying effects in the isolated vessel modelhas been demonstrated using a pilot study of 6 sham exposed and 18 highintensity focused ultrasound exposed vessels, with or without intraluminalcontrast agent (SonoVue) within the vessels.
(Some figures may appear in colour only in the online journal)
Introduction
The effect of ultrasound exposure on blood vessels is of importance both for therapeuticultrasound and for the safety of diagnostic ultrasound. Diagnostic ultrasound (US) is a widelyused real-time imaging tool whose utility has been improved by the use of microbubbleultrasound contrast agents (UCAs). Microbubbles have also found application in ultrasoundtherapy (Unger et al 2002). At sufficiently high peak negative pressure (PNP) amplitudes, theyundergo oscillations that may be stable, or may be violently destructive, leading to bubble
wall disruption, jetting and the creation of new gas filled bubbles (Miller and Thomas 1995).Both processes can influence surrounding cells by increasing their membrane permeability.This can facilitate gene and drug delivery (e.g. Christiansen et al (2003), van Wamel et al(2006)). Ultrasound activation of microbubbles may lead to lethal effects which should beavoided in diagnosis, but could be advantageous in therapy. Ultrasound induced contrast agentbioeffects have therefore been studied in many in-vitro and in-vivo models in order to assesssafe regimes for their use. Detailed reviews of bioeffects from ultrasound exposure of UCAhave been published (Miller 2007a, 2007b, Dalecki 2007, ter Haar 2009).
Ultrasound bioeffects are studied in three different experimental model types: in-vitrocell suspensions or cell monolayers, ex-vivo isolated animal tissue and in-vivo models. Cellsuspensions lack attachment and direct communication between cells. Assessment of theirresponse to an ultrasound exposure in suspension culture is difficult because the positionof each cell in the field, and hence its exposure level, is unknown. Whilst cell monolayersovercome some of these problems, they usually consist of a single cell type and may be subjectto reflections of the sound beam from the culture dish or water/air interface (Leskinen andHynynen 2012). The attachment of cells in monolayers differs from intercellular binding foundin living organisms (Cretel et al 2008). Ex-vivo and in-vivo models are superior in terms ofphysiology, cellular structure and function, but assessment of in-situ pressure values is difficult.Small animal models present problems of scaling, and exposure, not only of the volume ofinterest, but also of surrounding ‘normal’ tissue. In ex-vivo and in-vivo models, many cell typeslie within the ultrasound field and assessment of interactions between these and ultrasound( ± UCAs) can be complicated. The concentration of contrast agent in the exposure volume isusually not accurately known. Vascular response in-vivo results from complicated interactionsbetween the different cell types present in the wall with the nerve endings, metabolites andhormones in their surroundings. This complexity is absent when using isolated vessel models.
Isolated blood vessels have seldom been used for ultrasound exposure studies. Hendersonet al (2010) used excised, unperfused ovine veins (diameter: 1–5 mm), overlain bycombinations of skin, fat and muscle, for study of HIFU induced ablation of the vesselwall. Another approach has been to use the microvessels of the elastic, semi-transparentmembrane found in mammalian intestines (Caskey et al 2007, Chen et al 2010b, 2011) orcremaster muscle (Samuel et al 2009, Schneider et al 2012). A similar structure is providedby the chorioallantoic chick embryo model used by Stieger et al (2007) for the study offluorescein isothiocyanate (FITC)-dextran molecule extravasation. Chen et al (2011) observeddirect interaction between microbubbles (<10 μm diameter, single or in a small group) andendothelial cells (ECs) in excised rat mesenteric microvessels (<100 μm). Vessels exposed tosingle 1 MHz pulses (2 μs at 0.8–4 MPa) exhibited distension and invagination at the positionof the bubble. Invagination was associated with EC separation (Chen et al 2010a, 2010b).
Martin et al (2012) investigated changes in wall tension and mesenteric vessel diameterin isolated equine arteries (external diameter: 0.5–8 mm). The temperature at the focus ofthe acoustic field where the vessel was situated was measured using a soft-tissue thermal testobject, but no attempt was made to monitor acoustic cavitation. Vessels were submerged in a37 ◦C Krebs–Ringer solution and exposed to a weakly focused 3 MHz ultrasound beam (4 minexposure of 0.12–0.18 MPa PNP). A vessel wall stress of 0.04 ± 0.03 mN mm−2 was foundafter exposure to the highest PNP. A mesenteric artery, with intraluminal flow, was located atthe bottom of a 37 ◦C Krebs–Ringer filled tank and imaged with an inverted microscope duringexposure from above for 4 min at 3 MHz 0.18 MPa PNP. Similarly to the results from equinearteries, ultrasound induced constriction was seen in large mesenteric arteries but not in smallones, leading the authors to attribute this result to a lack of temperature sensitive channels inthese vessels (Martin et al 2012). Isolated mouse aortas, containing circulating drug-bearing
Ultrasound exposure of isolated blood vessels 2283
liposomes in intraluminal buffer, have been used in a study of drug delivery (Hitchcock et al2010). This system allowed control of the intraluminal pressure, blood flow and concentrationof liposomes being delivered to the aorta.
High intensity focused ultrasound (HIFU) has been used to occlude blood vessels andcreate controlled damage to soft tissue in animal models (Hynynen et al 1996, Rivens et al 1999,Ichihara et al 2007). The effects of in-vivo HIFU exposure of blood vessels containing contrastagents are not well understood. Enhanced tumour ablation has been demonstrated in animalmodels. Kaneko et al (2005) found larger lesions in animals given Levovist (300 mg ml−1)than in saline injected animals, following exposure to 2.18 MHz HIFU (ISPTA = 420 W cm–2).Similar studies by Luo et al (2007) showed an increase in liver lesion size after 2 s HIFUexposure of 600 W total acoustic power in the presence of SonoVue. Zderic et al (2006)reported a reduction in the time required to induce haemostasis in haemorrhaging rabbit liver(using 6800 W cm–2 in-situ intensity at 5.5 MHz HIFU) from 70 ± 23 s without Optison to59 ± 23 s when it was injected intravenously.
In this paper we describe the development of an experimental system in which an isolatedrat mesenteric artery can be exposed to a well characterized ultrasound pressure field in thepresence, or absence, of intraluminal UCA, with simultaneous cavitation monitoring. Thismodel, which is widely accepted for the study of vasculature in other areas of research (vanBavel et al 1991, 1998, Pistea et al 2005, Bakker et al 2008), has been modified here for usein a novel vessel chamber whose design is based on a standard, pressure myograph system,adapted for vessel ultrasound exposure by the introduction of an acoustic window.
A standard system for the delivery of HIFU exposures with simultaneous cavitationdetection (McLaughlan et al 2010) has been modified for use here. Cavitation monitoringwas performed using a fibre-optic hydrophone (FOH) sensor. The vessel appearance andbehaviour was monitored through a microscope before, during and after exposure. Fluorescentmicroscopy was used to study vascular permeability, while functionality was investigated usingvitality tests for assessment of endothelial and smooth muscle cell (SMC) response. Whilethis experimental arrangement overcomes many of the limitations of other models, it is notcompletely representative of in-vivo exposures since the vessel lacks surrounding tissue, and istherefore susceptible to effects occurring at the outer vessel wall/feeding buffer interface. Thispaper reports preliminary studies of the behaviour of 6 sham and 18 HIFU exposed vessels,some containing UCA, carried out to assess the utility of this experimental model.
Materials and methods
Overall system and experimental protocol
The experimental system is shown schematically in figure 1 and the experimental protocol isoutlined in figure 2.
The main role of the vessel chamber (figure 3) was to isolate a vessel, immersed in300 ml of temperature controlled (37 ± 1 ◦C) feeding buffer, from the tank containingthe degassed water required for ultrasound propagation. The extraluminal buffer temperaturewas maintained by thermal conduction from the lower water bath. The open buffer surfacewas covered with Melinex membrane to reduce cooling, evaporation and ultrasound induceddisruption as discussed later.
An ultrasonically transparent Melinex membrane (5 cm in diameter, 19 μm thick (PMH980, HiFi Industrial Film Ltd, Stevenage, UK)) provided an acoustic window in the base of thechamber. A planar-tipped calibrated FOH, with a 140 μm diameter fibre and a 10 μm activesensor at its tip (Precision Acoustics, UK), was used for acoustic pressure measurements.
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Figure 1. Schematic diagram of the experimental system designed for maintenance of a viableblood vessel in the vessel chamber, its exposure to therapeutic (or imaging) ultrasound withsimultaneous detection of cavitation and observation of the vessel behaviour.
The 3D micropositioner (Newport Spectra-Physics Ltd, Didcot, Oxfordshire, UK), on whichthe FOH was mounted, was attached to the side wall of the vessel chamber. This allowed (i)beam plotting within the vessel chamber, (ii) spatial-peak intensity calibration as a functionof HIFU drive level, (iii) precise localization of the vessel within the HIFU focal region and(iv) cavitation detection during ultrasound exposures.
Vessel isolation, preparation and maintenance
Female Sprague–Dawley rats (250 ± 40 g) were sacrificed using 100% CO2, in accordancewith UK Home Office guidelines. The intestine was isolated, immediately stretched ona Sylgard 128 covered petri dish (World Precision Instruments, Sarasota, FL, USA) andimmersed in 4 ◦C MOPS buffer (in mMol l–1: 145.0 NaCl, 4.7 KCl, 2.40 MgSO4, 2.65 CaCl2,1.38 NaH2PO4, 3.0 MOPS, 5.0 glucose and 2.0 pyruvate, at pH = 7.35 (all components suppliedby Sigma-Aldrich Company Ltd Dorset, UK)). Using a dissection microscope (OPMI 1-FC,Zeiss, Germany), a ‘1st order’ branch artery (figure 3(c)), of external diameter 300–600 μmwas cleaned of attached fat tissue and excised from the mesentery in readiness for mountingin the vessel chamber.
Two 125 μm diameter tipped glass micropipettes (Living Systems Instrumentation,Vermont, USA), to which the isolated vessel would be tied using 10 μm diameter nylon thread,were mounted within the vessel chamber. Prior to vessel cannulation, both micropipettes wereattached, using rubber O-rings, to slide-rails which allowed manual positioning. One pipettewas connected to the rail by a positioner that provided fine localization (in 50 μm steps)over a 1 cm range. This arrangement allowed vessel length adjustment after cannulation. Themicropipettes were each connected by polythene tubing (0.86 ID, 1.27 OD, Portex Fine Bore,
Ultrasound exposure of isolated blood vessels 2285
Animal preparation and mesenteric artery isolation
(1-2 h)
Fluoroscopy imaging for leakage &
incubation at 37oC (1 h)
Second vitality test image acquisition (20 min)
First vitality test image acquisition (20 min)
Buffer change and 30 min incubation at 37oC
Normalisation vessel diameter measurement
(8 min)
Ultrasound exposure followed by observation
(15 min)
Blood vessel cannulation and UCA infusion if required
(1-2 h)
Preservation for histology(5 min)
Figure 2. Schematic diagram showing the experimental protocol used in this study.
Smith Medical Int. Ltd, Hythe, UK) to a 25 ml reservoir containing MOPS buffer plus the0.5% albumin necessary to maintain appropriate osmotic pressure and 106 M of 70 kDa FITC-dextran (Sigma-Aldrich Company Ltd, Dorset, UK) used for the fluorescence microscopystudies described below. Equal physiological (80 ± 1 mmHg) pressures, generated usingelectro-pneumatic pressure transducers (T5200, Fairchild, Winston-Salem, NC, USA) builtinto a custom-made controller, were initially applied to these bottles in order to fill the tubesand micropipettes with buffer and to maintain pressurization of the cannulated vessel withoutflow (which could complicate interpretation of HIFU induced bioeffects).
All pipette-mounted arteries were carefully checked by microscopic inspection at40 × magnification for leakage of fluorescence (FITC) into the extraluminal buffer whichwould indicate cannulation damage or the presence of severed side branches. Leaking vesselswere discarded.
Imaging and analysis of blood vessel diameter
Extensive microscope measurements of vessel diameter were required to verify whetherstandard blood vessel functionality assessment methods could be used for monitoringultrasound induced vascular effects. Images of isolated vessels were obtained before, duringand after HIFU (and sham) exposure using an upright fluorescent material science microscope(BXFM, Olympus, Germany), with a 4 × magnification 17 mm working distance objective(UPlanFLN). This was chosen for its ability to image the entire width of samples mounted in
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(a)
(b)
(c)(d)
Figure 3. Vessel chamber (25 × 10 × 3 cm3). (a) Top view of the whole chamber. (b) Magnifiedview of the centre of the chamber showing the micropipettes and FOH sensor located above thecircular acoustic window. (c) A rat intestine stretched on a Sylgard 128 covered petri dish with a 1storder vessel indicated. (d) An isolated artery tied to micropipettes and at an intraluminal pressurizeof 80 mmHg.
the vessel chamber when positioned above the buffer surface, avoiding interaction with theultrasound beam.
Intense light excitation of FITC may cause adverse effects in blood vessels, (Herrmann1983). The settings used for fluorescence imaging were therefore: 120 ms illumination time(the shortest time permitted by the software); 500 ms time cycle (the minimum practical); lightintensity: 1.7% (the lowest possible) for ‘T-Red’ and 100% for ‘FITC’. The FITC imaging
Ultrasound exposure of isolated blood vessels 2287
light level was dictated by the relatively low concentration of FITC-dextran in the intraluminalbuffer (10−6 M, chosen to minimize the effects of dextran binding to some receptors on theendothelium, and of osmotic effects resulting from replacement of water by dextran molecules).This concentration has been recommended for isolated vessel studies (e.g. van Haaren et al(2003)). As sham exposed vessels underwent the same imaging protocol, any adverse effectsarising from light exposure should have been seen.
The microscope was aligned so that the region of the vessel to undergo ultrasound exposurewas in the centre of the captured image. This was achieved by moving the FOH tip adjacent tothe vessel wall and rotating the microscope body so that both were displayed in the acquisitionwindow.
Normalization of vessel diameter. In order to compare vessels of different diameters it wasnecessary to perform a normalization (van Bavel et al 1991). The diameter used for thiswas determined at the start of each experiment, prior to the vitality test, by increasing theintraluminal pressure from 20 to 120 mmHg in 30 s, 10 mmHg steps, and then lowering itback to 80 mmHg. The time averaged diameter at the final pressure, commonly referred to asd0, was used for subsequent normalization (Sun et al 1992, van Bavel et al 1998). Calcium-ion-free medium is usually used for this test to avoid activation of the SMCs. However it hasbeen shown by Sun et al (1992) that the difference between first branch rat mesenteric arteryconstriction in normal and calcium-free media is minimal. Thus, in order to avoid an additionalbuffer change, the assessment was here performed in normal MOPS buffer (Sun et al 1992).
Blood vessel vitality tests. Vitality testing, a standard technique used in many cannulatedvessel studies, exploits the activation of signalling pathways by exposing vessels tovasoconstricting or relaxing drugs, and measuring their response. Vasoconstriction can beinduced by the binding of phenylephrine (Phe) to SMCs, while vasorelaxation can be activatedby acetylcholine (Ach) attachment to receptors on ECs. Phe is an α1-adrenergic receptoragonist, which, similarly to adrenalin, activates sympathetic responses such as the constrictionof arterial wall smooth muscle (Nishimura et al 1989). Ach activates endothelial nitric oxidesynthase, the production of which affects other cellular transmitters, leading to vasodilation(dilation of the blood vessel’s SMCs (Furchgott et al 1987)). Addition of these drugs in aspecific sequence will here be referred to as a ‘vitality test’ (vt1 being the test performedbefore ultrasound (or sham) exposure, and vt2 that undertaken afterwards).
Vitality tests started with 5 min of vessel incubation in Phe (10−6 M), to inducemaximal constriction of a normally functioning vessel, followed by incubation at increasingconcentrations of the vasodilator Ach (10−8, 10−7, 10−6, 10−5 M), each for a 3 min observationperiod. The concentrations used have already been proven to affect rat mesenteric arteries andare accepted as standard (e.g. Looft-Wilson and Gisolfi (2000), Hwa et al (1994), McIntyreet al (1998)). Each drug was pipetted into the extraluminal buffer, with the infusion takingplace less than 10 mm from the cannulated vessel. Lack of response to vt1 (indicating damage
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during isolation and cannulation) resulted in the vessel being discarded. Next, the extraluminalbuffer was removed and replaced with fresh MOPS followed by 30 min incubation, allowingthe buffer to reach 37 ◦C. The second vitality test (vt2) was performed 15 min after ultrasound(or sham) exposure.
Blood vessel functionality analysis. In order to compare the functionality of different vesselsbefore and after HIFU exposure, normalized vessel diameters were used to calculate twocommonly applied vascular response parameters (Hwa et al 1994, Kimura et al 2002, Earleyet al 2004): percentage constriction (equation (1)) and percentage relaxation (equation (2)).These require determination of ‘StartDRUG’, ‘Phe’ and ‘Ach’ parameters, defined as the vesseldiameters measured just before addition of drugs, and after Phe and Ach administration,respectively.% constriction:
StartDRUG − Phe
StartDRUG× 100%. (1)
% relaxation:Ach − Phe
StartDRUG − Phe× 100%. (2)
Average ‘StartDRUG’, ‘Phe’ induced minimum and ‘Ach’ induced maximum vessel diameterswere calculated as the mean value over 2–5 s for which the diameter has changed only withinmeasurement uncertainties (van Bavel et al 1991).
Blood vessel permeability assessment
Changes in vessel permeability resulting from ultrasound exposure were investigated byobservation of the fluorescent intensity of FITC labelled dextran (hydrodynamic radius ∼6.5 nm) mixed with intraluminal buffer at a concentration of 10−6 M. Fluorescence microscopyimages were acquired during vt1 in order to monitor baseline appearance and then during theHIFU exposure and vt2 periods to look for leakage. This dye, whose emission wavelength is518 nm, was chosen as its size means that it remains in solution inside intact vessels (Nakamuraand Wayland 1975). Its presence in the extraluminal buffer and/or uptake in the vessel wallwould therefore indicate increased vessel permeability.
Histology
Histological examination was carried out on vessels fixed immediately after the study end.In order to avoid vessel collapse during fixation (performed immediately after completion
of vt2), the extraluminal buffer in the vessel chamber was replaced with 10% normal bufferedformalin (Pioneer Research Chemicals Ltd, Colchester, UK) for 5 min whilst the vesselsremained attached to the micropipettes and pressurized to 80 mmHg. Samples were thenremoved and fixed for at least a further 3 days before being embedded in wax blocks forcutting into 5 μm thick tissue sections and mounting on glass slides. In order to identifyregions of ultrasound induced damage, the whole length of the sample was cut in orthogonalcross-section and then a few (usually five) sections at 200 μm intervals were chosen forstaining with Haematoxylin and Eosin (H&E) (Pioneer Research Chemicals Ltd, Colchester,UK) and were examined using a BX61 microscope (Olympus, Germany). These dyes staincell nuclei blue and other cellular structures pink. The appearance of sham and ultrasoundexposed vessel sections was compared.
Ultrasound exposure of isolated blood vessels 2289
Ultrasound exposure with simultaneous cavitation detection
A 1.7 MHz HIFU transducer (Siemens A2-2, focal length: 7.3 cm, diameter: 5.6 cm) was used.It was immersed in 10 μm filtered degassed (5.66 ± 0.29 mg l–1 dissolved oxygen) tap water at37 ◦C as shown in figure 1. The transducer was driven by an amplified signal from a waveformgenerator (33120A, 15 MHz function generator, Agilent Technologies, CA, USA), which wastriggered by a timer set to 5.0 s. A ‘Pick-off’ box (which allowed drive voltage monitoringas described below) and a 50 � impedance matching circuit were connected after the poweramplifier (ENI A500 amplifier). Ultrasound field measurements were made using a FOH probemounted inside the vessel chamber on a micropositioner, as shown in figure 3, which allowedlocalization of the focal peak to within ± 1 μm. Hydrophone voltage measurements wererecorded on an 8-bit oscilloscope (WaveRunner 64Xi, LeCroy Corporation, NY, USA), andwere converted to pressure using the frequency dependent sensitivity calibration provided witheach probe.
Vessel targeting. Prior to sample mounting, the FOH tip was positioned, under microscopeimaging guidance, between the pipette tips at the experimental position for the centre of thevessel, and the HIFU focal peak was moved to coincide with this. The mean uncertainty inalignment with a vessel was ± 150 μm, due to the necessity of removing the vessel chamberfrom the degassed water tank after focal alignment, in order to allow sufficient access forvessel mounting on the micropipettes, and its subsequent replacement.
HIFU calibration. As predicted by a simple field model, preliminary beam plotting studies,using the FOH sensor mounted within the vessel chamber, carried out to establish the angleat which the amplitude of standing waves created by reflection of the ultrasound field atthe buffer/air interface above the chamber was minimum, showed that the transducer shouldbe angled at approximately 22.5◦ to the vertical (figures 4(a) and (b)). The maximum availableangle used for this transducer in the tank was 30◦. Long pulse lengths were used for theseplots in order to accentuate any standing wave formation.
The acoustic field along the direction of the vessel’s length was plotted using 2.9 MPaPNP (3.5 MPa peak positive pressure, PPP), 20 cycle, 100 Hz pulse repetition frequency. Thiswas chosen to be within the FOH linear pressure response range (PNP � 4 MPa and PPP �7.5 MPa (Morris et al 2009)), but was lower than levels used in vessel studies. The plottedvalues of pressure at each position were obtained by averaging 100 waveforms. Pressurevalues were calculated by calibrating the fibre against a 0.4 mm PVDF membrane hydrophone(Precision Acoustics, UK) using the substitution technique (Smith and Bacon 1990) andfrequency deconvolution at the first four harmonics. The beam profile is shown in figure 4(c).The uncertainty associated with this measurement was estimated to be ∼15%, based on themeasurement uncertainty of the reference hydrophone (7%) and the ability to position bothhydrophones at exactly the same location in the acoustic field.
The focal pressures used for vessel exposures were obtained from free-field measurementsusing the 0.4 mm membrane hydrophone. The PNP and corresponding PPP chosen were 4.3(9.1) MPa and 5.0 (13.5) MPa (uncertainty ∼ 10%). The pressure values were obtained usingfrequency deconvolution up to 40 MHz.
Cavitation detection. Acoustic cavitation and potential tissue water boiling were monitoredusing a suite of detection hardware and techniques based on those developed by McLaughlanet al (2010). A 10 μm diameter FOH was used as a passive cavitation detector (PCD). DuringHIFU exposures, the fibre tip was placed 3 mm away laterally from the previously identified
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(a)
(b)
(c)
Figure 4. (a) Measurements made with the transducer pointing vertically upward (0◦). (b) Thevertical plot repeated with the transducer at 22.5◦ to the vertical. The longitudinal ultrasound profileswere generated using 200 cycles and 50 Hz pulse repetition rate. The FOH was moved vertically in40 μm steps, starting from the bottom of the chamber, towards the buffer/air interface. (c) LateralHIFU beam profile, obtained in the direction along the cannulated vessel length, measured withinthe vessel chamber using a FOH moved in 100 μm steps. Pressure FWHM: 1.6 ± 0.1 mm (PPP);1.9 ± 0.1 mm (PNP). These are wider than the vessel diameters used in this study (0.3 to 0.6 mm).The error bars represent the SD of three measurements.
Ultrasound exposure of isolated blood vessels 2291
position of the focus, to provide a compromise between maximizing sensor sensitivity tobubble emissions and avoiding sensor damage due to acoustic cavitation. The signal from theFOH was passed through a 1.7 MHz notch filter (F5181, Allen Avionics Inc., NY, USA) toremove the drive frequency, and was then amplified using a low noise 20 dB preamplifier (7866,0.1 to 30 MHz bandwidth, Advanced Receiver Research, Burlington, CT, USA). Data wereacquired continuously during 5 s exposures, and for the 2 s immediately afterwards to providebaseline noise data. For sham exposed vessels, data were collected for 7 s with the transducerdisconnected from the drive system, but with the power amplifier on. A second, simultaneous,cavitation detection technique monitored the HIFU drive voltage to detect fluctuations. Thefluctuations of up to 0.3 mVRMS which were observed in sham exposure experiments (n = 43)were considered to arise from noise. Therefore fluctuations greater than 0.3 mVRMS in drivevoltage were assumed to be indicative of cavitation activity.
FOH and drive voltage signals were sampled at 20 MHz using two channels of a highspeed 8-bit data acquisition card (MI.2031, Spectrum Systementwicklung Microelectronic,Germany) mounted in a desktop computer (P4SCT, SuperMicro Computer Inc., CA, USA) toprovide a useable ‘bandwidth’ of 0.5 to 10 MHz. A MatLab script was used to process both setsof recorded data. Briefly, raw binary FOH data were split into 2800 segments per second, anda fast Fourier transform was calculated for each segment. For this study, the total frequency-integrated RMS voltage of broadband signals in the range 7–8 MHz was plotted for each timepoint. Such broadband signals are indicative of inertial cavitation activity (McLaughlan et al2010). For drive voltage, the RMS value was calculated for each of 2800 segments per second.
As a third simultaneous cavitation detection technique, a microphone (CHK00627,Partridge Electronics, UK) was used to detect audible frequency emissions (2–20 kHz) whensubmerged in the vessel chamber ∼5 cm from the sample and the HIFU focus. Emissionsdetected were sampled with 16-bit resolution at a rate of 44.8 kHz using Smart PC Recordersoftware (v2.5) (free online), and then processed using a MatLab code which converted thedata into frequency spectra (McLaughlan et al 2010).
The criterion used to decide whether acoustic cavitation had occurred during a 5 s exposurewas that a broadband signal was detected over a minimum of two data points that were above thenoise baseline, set at eight standard deviations (SD) of the average of the noise. Average noisewas calculated using the full 2 s of data acquired after exposures were complete. This criterionwas identified during preliminary experiments and was chosen to minimize the number ofsham exposures in which false positive cavitation detection occurred. Audible emissions weredefined by the appearance of peaks that were not detected whilst the HIFU transducer wasinactive.
Experimental protocol
Twenty four intact rat mesenteric arteries were used to test the utility of the experimentalsystem described above. Arteries were isolated and cannulated as summarized in figure 2.SonoVue microbubble contrast agent (Bracco Diagnostics Inc., Italy) was prepared accordingto the manufacturer’s instructions and, for some vessels, added to the intraluminal buffer at aconcentration of 107 bubbles per ml and infused into the lumen during vessel cannulation.
Intact vessels were incubated for 1 h at 37 ◦C prior to assessment of d0. Their response toAch and Phe was then tested, thus providing initial vitality test (vt1) data. After external MOPSbuffer replacement, the vessels were incubated for a further 30 min at 37 ◦C before HIFUexposure. Single 5 s tone bursts of 1.7 MHz HIFU at one of three different PNP: 0 (shamexposure), 4.3 and 5.0 MPa were used, each with and without intraluminal UCA, to yieldsix different exposure conditions. Sham exposed vessels were used to check for changes in
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vessel response due to repeated vitality tests, extraluminal buffer changes and use of excitationlight for fluorescence imaging. Following HIFU exposure, the repeat vitality test, vt2, wasperformed. Finally, vessels were preserved for histological observation.
Uncertainties presented in this paper represent the SD of a series of measurements. Inorder to compare differences in mean percentage relaxation or percentage constriction of thesame experimental group (i.e. all vessels containing the same microbubble concentration andexposed to the same ultrasound pressure) at vt1 and vt2, a paired two-sample for means t-test(Analysis ToolPak, Excel 2007) was used.
Results
Vessel isolation, preparation and maintenance
Isolation of each rat mesenteric artery from the intestine took 15–30 min. Preparation of fourvessels was required in order to guarantee that there would be one which did not leak, andhad a ‘normal’ vt1 response (i.e. Phe constriction fully reversed by Ach addition). The finalobstacle to successful completion of an experiment was that at the highest HIFU exposure(5.0 MPa PNP) some vessels became detached from at least one of the cannulae. A total of24 useable datasets were obtained for 6 sham exposures (3 with UCA) and 18 HIFU exposedvessels (15 with UCA).
Imaging and analysis of blood vessel diameter
Microscopy imaging data were obtained during d0 assessment, during vt1 and vt2 and before,during and after HIFU (or sham) exposure. The radiation force associated with HIFU exposuresof 4.3 and 5.0 MPa PNP ‘pushed’ vessels out of the focal plane and field of view of themicroscope objective, respectively, making them impossible to image during exposure.
For 8 of the 24 vessels used in this study, ‘vasomotion’ (spontaneous vessel oscillationof varying frequency (van Bavel et al 1991) was observed. This usually started after Pheadministration and ended after the first dose of Ach. Since vasomotion could affect vesselresponse to drugs, it was important to acquire baseline data to minimize the effects of theseoscillations on the vessel diameter measurements.
Normalization of vessel diameter. The response of vessels to incremented static intraluminalpressures up to 120 mmHg followed by a return to 80 mmHg was studied. The vesselshad diameters in the range of 400–560 μm. These values of d0 were used to normalize allsubsequent diameter data on a per vessel basis.
Blood vessel functionality analysis. For ultrasonically exposed vessels, the position ofmaximum HIFU induced constriction was identified by finding the time point at which thisoccurred, and then averaging five adjacent diameter measurements separated by 16 μm atthe narrowest diameter. For sham exposed vessels, diameter measurements acquired at fivepositions with 16 μm separation at the image centre were averaged. The uncertainty in averagedvessel diameter was taken as the inherent measurement uncertainty of ± 4 μm. The largestuncertainty in normalized diameter was calculated to be ± 0.04. This worst case value hasbeen used to indicate the precision of all normalized diameters.
Plots such as that shown in figure 5, which demonstrate normalized vessel diameterbehaviour during vitality tests obtained before and after HIFU exposure, were used toprovide the required normalized vessel diameters for calculation of percentage constriction
Ultrasound exposure of isolated blood vessels 2293
Figure 5. A representative example of the response of a vessel containing SonoVue contrast agent(107 bubbles ml−1) to Phe (10−6 M) followed by increasing concentrations of Ach (10−8, 10−7,10−6 and 10−5 M) before (first vitality test: -�) and after (second vitality test: - • ) 5.0 MPa PNP,5 s HIFU exposure. The vertical lines show the time of addition of each drug to the extraluminalbuffer. The horizontal dashed lines indicate the periods over which the normalized diameter wasaveraged in order to minimize the impact of vasomotion. The arrows show periods of stable vesseldiameter (within the measurement uncertainty of ± 0.04) during which the average was obtainedover 2–5 s.
Table 1. Normalized vessel diameters obtained during the second vitality test: before any drugadministration (StartDRUG), after Phe administration and with increasing doses of Ach (Ach1 to 4)calculated using three different techniques for a single vessel exposed to 5.0 MPa with intraluminalcontrast (107 bubbles ml−1). The uncertainty in the averaged values is the SD. Peak values aregiven with measurement uncertainty. ∗ indicates vasomotion over the period used for averagingcalculations.
vt2 data for a vessel containing UCA exposed to 5.0 MPa PNP
(equation (1)) and percentage relaxation (equation (2)). Firstly the instantaneous peakconstricted or relaxed normalized diameter after each drug administration was determined.Secondly the ‘average peak’ parameters were determined either by time averaging over a2–5 s period during which they changed only within uncertainties ( ± 0.04) or, in a few casesin which vasomotion occurred, the data were averaged over a complete number of vasomotioncycles. Thirdly, the normalized ‘steady-state’ diameter was obtained using the time averagingoptions described above, but at a time when either an apparent steady-state had been reachedor, if this was not possible, immediately prior to the addition of the next dose of drug.
The results for the different techniques are summarized in tables 1 and 2. Table 1shows an example of normalized vessel diameters acquired for a single vessel containing
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Table 2. Averaged normalized vessel diameters obtained before any drug administration(StartDRUG), after Phe and with increasing doses of Ach (Ach1 to 4) quantified using three differenttechniques during the first vitality test. The values are averaged for the 24 vessels used in this study.Uncertainty is SD.
107 bubbles ml−1, exposed to 5.0 MPa. This vessel was chosen to illustrate the effect thatvasomotion after Phe administration, and an altered response to Ach due to HIFU exposure(figure 5), can have on the different methods of quantifying normalized vessel diameter.The peak and average peak diameters after Ach induced vessel relaxation agreed withinuncertainties whereas the average steady-state values were smaller (as would be expected),resulting in final differences in percentage relaxation which would suggest better, or slightlyworse, EC response for peak and steady-state methods, respectively. However all threemeasurement approaches show that the vessel failed to relax back to its StartDRUG size.Vasomotion resulted in average and steady-state Phe diameters larger than the instantaneouspeak, with a significantly greater uncertainty in the data. Thus, use of the averaged values wouldresult in a smaller, and less precisely known, percentage constriction. However, taking the peakPhe diameter, the minimum value found in the vasomotion oscillation may be misleading. Inthis particular example, the Phe diameter values agreed within uncertainties. Peak diametersmay suggest better than, and steady-state diameters may suggest worse than, true vascularfunctionality.
Table 2 shows that peak diameters calculated from vt1 assessments using instantaneousand averaging methods were comparable for all Ach concentrations, with average steady-statevalues being marginally smaller, as expected, except after Ach4 where they were the samefor all three methods. Vasomotion occurred mostly after Phe and before Ach administration(n = 7/24) at vt1, and averaging over 24 vessels either ignoring (i.e. only using instantaneouspeak diameters) or including (i.e. using either time averaged peak or steady-state values)vasomotion gave diameter values that agree within uncertainties (table 2). No significantdifference was found between any of the techniques for quantifying ‘StartDRUG’, ‘Phe’ and‘Ach4’ diameters. Vessel diameter was always found to increase for rising Ach concentrations,whichever analysis technique was used.
Since there was little difference between the three methods, the standard method of peakvalue averaging was chosen for calculation of percentage constriction and relaxation.
HIFU (with no extraluminal drug present) was found to induce constriction which startedduring exposure and increased over time, followed by eventual dilation. An example of thisis shown in figure 6. Normalized vessel diameter change was used to assess the response toexposure in a way similar to the assessment of vt1 and vt2. A ‘StartHIFU’ normalized vesseldiameter prior to HIFU was obtained by averaging over 2–5 s just before the exposure, andthe minimum normalized diameter within 15 min of exposure (HIFUmin) was obtained byaveraging over 2–5 s at the peak constriction. The HIFU induced percentage constriction wascalculated using equation (3).HIFU induced % constriction:
StartHIFU − HIFUmin
StartHIFU× 100%. (3)
Ultrasound exposure of isolated blood vessels 2295
Figure 6. An example of changes in normalized vessel diameter with time after HIFU exposure fora UCA containing vessel exposed to 5.0 MPa for 5 s. During exposure, the vessel diameter could notbe measured. Afterwards, there was an initial constriction with eventual relaxation back to roughlythe start diameter. The arrows indicate 2–5 s periods of vessel size stability (within measurementuncertainty) over which diameter data were averaged. Normalized diameter uncertainty is 0.04.
HIFU effects on isolated vessels. The behaviour of vessels exposed to one of six conditionswas studied: PNP of 0, 4.3 or 5.0 MPa each in the presence (at 107 bubbles per ml), or absence,of intraluminal contrast agent.
Figure 7(a) shows the Phe induced percentage constriction of vessels for vt1 and vt2.Here the vt1 data were divided according to subsequent exposure conditions. Figure 7(a)shows that Phe administration during vt1 caused all 24 vessels to constrict by 47.2% ± 4.3%(average ± SD of all data). After HIFU exposure, some vessels (n = 10) exhibited behavioursimilar to that observed during vt1. Only vessels with 107 bubbles ml−1 exposed to 4.3 MPa(n = 3) or 5.0 MPa (n = 8) showed a reduced response to Phe with average normalizeddiameters of 0.62 ± 0.06 and 0.70 ± 0.16, respectively. Paired t-tests showed no statisticaldifference between vt2 and vt1 data for any experimental group, as shown in table 3.
Figure 7(b) compares the percentage relaxation from vt1 with that from vt2. Pre-exposure(vt1), vessels were constricted by Phe addition as discussed above, and expanded afteradministration of Ach4, to a size close to d0, the value used for normalization. The normalizeddiameter thus returned to approximately unity. Similar behaviour was found for sham exposedvessels from vt2. Vessels with or without UCA exposed to HIFU were characterized byreduced vessel diameter after Ach4 administration at vt2 compared to vt1, as reflected bya lower percentage relaxation than in sham exposed vessels, as shown in figure 7(b) andtable 3. A paired t-test showed that the observed difference between vt1 and vt2 percentagerelaxation was only statistically significant (p value < 0.05) for arteries exposed to 5.0 MPawith 107 bubbles ml−1 as shown in table 3.
HIFU exposure caused constriction of between 7% and 21%, which was significantlyless than that resulting from Phe administration (∼50%) during vt1 (table 3). The uncertaintyassociated with the calculation of HIFU induced percentage constriction, obtained usingerror propagation, was 7%, and therefore the constrictions observed in vessels with
2296 A Tokarczyk et al
(a)
(b)
Figure 7. Percentage normalized vessel diameter constriction (a) and relaxation (b) during vt1 andvt2 (i.e. before and after HIFU exposure to 0, 4.3 and 5.0 MPa negative pressure for 5 s in thepresence or absence of intraluminal SonoVue). The error bars represent SD.
Table 3. Fractional ratio of percentage constriction vt2 to percentage constriction vt1 (shownseparately in figure 7(a)) and fractional ratio of percentage relaxation vt2 to relaxation vt1 (shownseparately in figure 7(b)) for each HIFU experimental group in the presence or absence of ultrasoundcontrast (UCA). Paired t-test p values were used to compare the data within experimental groupsas shown. Fields highlighted in grey indicate experimental groups for which there is significantdifference between vt1 and vt2 data. Cavitation activity is indicated by BB—broadband emissions,DV—drive voltage fluctuations and/or AE—audible emissions for one vessel for each exposurecondition. The propagation of the uncertainties was calculated for each vessel.
0 MPa + 4.3 MPa + 5.0 MPa +0 MPa UCA 4.3 MPa 5.0 MPa UCA UCA
Constriction 0.94 ± 0.03 0.90 ± 0.10 0.75 ± 0.20 0.92 ± 0.20 0.70 ± 0.22 0.73 ± 0.35vt1/vt2 p = 0.23 p = 0.31 p = 0.21 p = 0.45 p = 0.18 p = 0.08
(n = 3) (n = 3) (n = 3) (n = 4) (n = 3) (n = 8)Relaxation 0.97 ± 0.01 1.01 ± 0.02 0.47 ± 0.24 0.80 ± 0.15 0.39 ± 0.31 0.36 ± 0.34vt1/vt2 p = 0.13 p = 0.32 p = 0.09 p = 0.10 p = 0.10 p = 0.001
Ultrasound exposure of isolated blood vessels 2297
(a)
(b)
(c)
(d)
(e)
Figure 8. Row A: examples of frequency-integrated broadband plots detected using the FOH inwhich cavitation events appear as peaks above the baseline noise level (horizontal line) in allexperimental groups except the sham exposed vessel. Row B: corresponding fluctuations found indrive voltage plots. Row C: audible frequency emissions recorded by the microphone; the line at 0 s(arrows) in some plots was an artefact caused by the HIFU being switched on. Row D: images ofthe fluorescence of the intraluminal FITC-dextran showing changes only in HIFU + UCA exposedvessels. Row E: H&E stained histology sections showing cellular structure with possible HIFUinduced damage demonstrated by discolouration of the vessel wall (circled), holes in tunica media(solid black arrows) and EC removal (dotted black arrows). Vessels were exposed to 5 s of 1.7 MHzHIFU at the PNP indicated in the column heading. UCA indicates the presence of intraluminalSonoVue at a concentration of 107 bubbles ml−1.
107 bubbles ml−1 or with no UCA after exposure to 4.3 or 5.0 MPa, respectively, areindistinguishable from zero within uncertainties.
Acoustic cavitation monitoring
The observed acoustic cavitation activity is summarized in figure 8. The simultaneouslyobtained broadband emission, drive voltage fluctuation and audible emission plots for 5 sexposures to 0, 4.3 and 5.0 MPa HIFU, with and without SonoVue contrast agent in theintraluminal buffer, and with the exception of audible emissions, for an additional 2 s after theHIFU was switched off, are shown. Corresponding fluorescent microscopy images and H&Estained sections are also shown. Sham exposed vessels behaved similarly with and withoutSonoVue.
The broadband baseline noise signal detected by the FOH in the absence of HIFU outputwas unstable and two sham exposed experiments showed that eight SD calculated over the
2298 A Tokarczyk et al
(a)
(b)
(c)
(d)
(e)
(f)
Figure 9. (a), (b) Frequency-integrated broadband, (c), (d) drive voltage fluctuation and (e), (f)audible emissions registered in MOPS buffer without UCA during 4.3 MPa (top row) and 5.0 MPa(bottom row) PNP 5 s exposure.
2 s period after exposure were needed to avoid false positive detection of cavitation. All datapoints above noise (line in figure 8(a)) indicated cavitation activity. These spikes were presentin all exposed experimental groups. Drive voltage fluctuations (figure 8(b)) greater than thosefound in sham exposed experiments (0.3 mVRMS) were observed in all exposed experimentalgroups and coincided approximately with most of the frequency-integrated broadband peaks.The audible frequency emissions (figure 8(c)) showed some peaks in the 10–20 kHz rangeonly during exposures of vessels at 4.3 MPa with UCA and 5.0 MPa (with and without UCA).Due to the difference in data sampling rates, fewer audio peaks were seen than broadbandemissions. Sham exposed vessels ± UCA showed no audio peaks.
Vessel permeability
Intraluminal FITC fluorescence was homogeneous in all vessels before HIFU exposure(figure 8(d-i)), with no evidence of leakage into the extraluminal buffer. Similar distributionswere seen in all HIFU exposed vessels in the absence of contrast agent (figure 8(d-ii) and(d-iii)). Where contrast was present during 4.3 and 5.0 MPa PNP exposures, hyperintensespots appeared in the vessel wall and there was leakage of fluorescent dye into the extraluminalbuffer (figure 8(d-iv) and (d-v)).
Histology
Figure 8(e) shows images of H&E stained sections. These were chosen to represent the mostabnormal appearances seen. The most striking difference between sham and exposed vesselsis their shape. Sham exposed samples had a uniform annular shape whereas exposed vesselswere usually elongated, with regions of their walls appearing thinned. H&E staining of thevessels containing contrast and exposed to 4.3 and 5.0 MPa PNP (figure 8(e-iv) and (e-v))showed reorganization of the nuclei and holes in the tunica media (solid black arrows). Inaddition, some nuclei were missing from ECs inside the lumen (dotted arrows). These vessels,
Ultrasound exposure of isolated blood vessels 2299
and the vessel exposed to 5.0 MPa without UCA, contained areas of less intense (pink) eosinstaining when compared to adjacent cells (circled in figure 8(e-iii)–(e-v)).
Discussion and conclusions
A novel experimental system designed to allow simultaneous ultrasound exposure, vesselmicroscopy and acoustic cavitation monitoring of an isolated rat mesenteric artery in atemperature controlled environment, followed by its fixation under normal physiologicalconditions has been described. Preliminary work has shown that it is suitable for study ofthe effect of HIFU on these vessels. The chamber allows both ultrasound exposure of vesselsand their imaging with an upright microscope, except during the period of HIFU exposurewhen radiation force may move the extraluminal buffer and/or cannulated vessels sufficientlyto cause the vessel to no longer be visible in the images. Vessels exposed to 4.3 MPa weremoved less than those exposed to 5.0 MPa, allowing acquisition of blurred images of theartery which were inadequate for diameter measurement. Thus the distance by which the4.3 MPa exposures stretched vessels could not have exceeded the depth of field of themicroscope objective (∼70 μm). However, as shown in table 3, HIFU exposure at 4.3 MPaPNP suggests greater vessel constriction than at 5.0 MPa PNP. We may speculate that vesselmovement has no, or only minor, effect on the constriction or that 5.0 MPa exposures move thevessel out of the beam, thus reducing exposure and resulting in less HIFU induced constriction.
In this study, the arteries had diameters in the range of 300–600 μm; however replacementof the micropipettes with cannulae which match the inner diameter would allow other vesselsizes to be studied. During cannulation both buffer and UCA (where required) were infusedinto the lumen. The microbubbles therefore stayed inside the vessel for ∼1.5 h prior to HIFUexposure. Recently it has been shown that SonoVue bubbles placed in a tube at 37 ◦C increasedin average diameter and had decreased stability compared to those at 20 ◦C (Mulvana et al2010). Therefore it would be beneficial to infuse microbubbles immediately before HIFUexposure, to ensure that their number and size are unaltered and more clinically relevant. Thiscould be achieved in this system by inducing flow by application of a pressure differencebetween the buffer containing reservoirs. However the timing, character (shear stress) andeffects of flow on the vessel must first be determined.
The small FOH was chosen as a sensor because of the limited space within the vesselchamber, but was found to have a number of other significant advantages: (i) its good spatialresolution allowed detailed ultrasound field measurements within the chamber, (ii) it allowedlocalization of the HIFU focal peak at a position centred between the micropipettes where thevessel was subsequently mounted and (iii) it facilitated acoustic cavitation detection duringHIFU exposures. Although the HIFU focus could be localized to within 1 μm, the vesselchamber had to be removed from the water tank after HIFU alignment to allow for vesselmounting, after which the chamber was re-mounted on the water tank wall. The quotedtargeting accuracy of 150 μm corresponds to half the diameter of the smallest vessel usedin these preliminary studies. Therefore, it would be advantageous to use vessels of 300 μmminimum diameter, as here, in all future studies.
The system was designed to allow vessel shape preservation during histological fixationby filling the chamber with formalin while the vessel remained pressurized to 80 mmHg.Sham exposed vessels had annular vessel structure which suggested that this technique wascapable of preserving vessel shape. Whilst the exposed vessels did not have a circular cross-section, there is currently insufficient evidence to know whether this is a consequence of vesselstretching during exposure, or of histological processing, due to tissue sectioning at an anglethat was not orthogonal to the vessel length, but in any case was only found in exposed vessels.
2300 A Tokarczyk et al
Pilot experiments have provided anecdotal information about the response of vessels toHIFU exposures with and without contrast agent, and allowed preliminary assessment of theprecision with which measurements can be made.
d0 was assessed prior to vt1 from change in diameter due to increasing pressure(20–120 mmHg). This is a technique commonly used for assessment of the passive vesselproperties i.e. those originating from the elastin and collagen content, rather than SMC activity(Brayden et al 1983). In future, this test will be repeated at the end of the experiment in orderto investigate effects of HIFU on elastin and collagen. Appropriate histological stains wouldprovide additional information about the structure of these components in the vessel wall.
Vasomotion was observed here in 8 out of 24 vessels. It usually occurred after Pheadministration and decayed with the first dose of Ach. Vasomotion has been investigated inmany vascular studies. Its activation by agonists (e.g. phenylephrine or adrenaline) has beenobserved, but other triggers have also been reported (Nilsson and Aalkjaer 2003, Aalkjær andNilsson 2005).
The averaged percentage relaxation for all vessels (n = 24) resulting from vt1 was 99.4%± 3.4%, indicating that Ach completely reversed the effects of Phe when the ECs werefunctionally normal (table 3). This demonstrates that the vessels tolerated the isolation andcannulation process well. Furthermore the sham exposed vessel vt2 behaviour (n = 6) suggeststhat vessel functionality can be maintained for at least 4 h. The reduction in the percentagerelaxation induced by Ach administration at vt2 indicates damage to ECs caused by 5 s HIFUexposures of 4.3 and 5.0 MPa PNP. The presence of SonoVue inside vessels appears likelyto amplify this damage as the percentage relaxation reduced from 79.3% ± 14.5% to 33.1%± 28.1% for 5 MPa PNP and from 46.6% ± 22.7% to 41.3% ± 33.1% for a 4.3 MPaPNP exposed vessel. This is likely to be due to contrast agents acting as cavitation nuclei andenhancing intraluminal inertial cavitation (Miller and Thomas 1995). Percentage constrictiondue to Phe administration was similar in all experimental groups.
HIFU exposure caused blood vessel constriction of between 7% and 21% (table 3).However, there was no obvious correlation observed between the exposure condition and thedegree of induced constriction. Studies of temperature sensitive channels localized in the nerveendings in the tunica media suggest a threshold of 42 ◦C for vascular effects (Huang et al2006, Scotland et al 2004). Significant temperature rises are unlikely here, because of heatdissipation in the extra- and intra-luminal buffer. It is therefore possible that constriction wasthe result of damage to SMCs due to the observed cavitation and/or vessel stretching due toradiation force.
Changes in vessel wall permeability were seen with intraluminal SonoVue after HIFUexposure at both 4.3 and 5.0 MPa PNP. The presence of bright spots in the vessel lumen ismost likely to be due to 70 kDa FITC-dextran molecules attaching to the extracellular matrix,and then filling the intercellular spaces after crossing a layer of damaged ECs. This size ofFITC-dextran molecules was chosen as they are larger than the average pore size of adherent(∼3 nm) and tight junctions (∼1 nm) in endothelium (Mehta and Malik 2006, Yuan and Rigor2010). No adverse effects of FITC excitation light on vessel functionality were found, asshown by the unaltered vitality of sham exposed vessels.
These observations agree with those reported by Stieger et al (2007) who observed leakagein chick embryo vessels (<55 μm) following ultrasound exposure with contrast agent. Electronmicroscopy revealed intra- and inter-cellular gaps in exposed vessel walls and extravasatederythrocytes and plasma. Although they did not state the size of the gaps, they were likely tobe >8 nm, the radius of the 150 kDa FITC-dextran used in their fluorescence studies. Theirobservations suggested the importance of direct contact between microbubbles and the vesselwall. We have shown that HIFU + UCA exposure is able to induce permeability changes in
Ultrasound exposure of isolated blood vessels 2301
large (300 to 600 μm) vessels, possibly because bubbles are pushed by radiation force, orfloat naturally, upwards into contact with the top wall of the vessel, and therefore ECs, beforeand/or during exposure.
H&E staining showed regions of lower eosin dye intensity, which may indicate the positionof the HIFU exposure along the vessel length. Areas of low eosin uptake were found in allvessels exposed in the presence of UCA and at 5.0 MPa without UCA. Similar discolourationhas been observed in HIFU lesions in liver in vivo (Rowland et al 1997, Chen et al 1999a,1999b). Additional histological or immunohistochemical staining would be useful to obtaininformation about the vascular structure and functionality of exposed vessels, in order tocorrelate these findings with the vitality test data.
The altered response to Ach at vt2, the HIFU induced constriction, structural damage andincreased permeability seen are likely to be caused by acoustic cavitation, whose occurrencehas been simultaneously monitored using broadband emissions, fluctuations in drive voltageand audible emissions. Broadband signals indicating inertial cavitation were observed in allHIFU exposed groups. The choice of noise level was dictated by the use of a FOH as aPCD. This was found to be less stable than the commonly used focused piezoelectric PCD(Bull et al 2011). Hence it was beneficial to use two other simultaneous cavitation detectionmethods in order to substantiate our findings. Unlike McLaughlan et al (2010), who onlyobserved power fluctuation or audible emissions in ex-vivo tissue when boiling occurred,here there were drive voltage fluctuations in all HIFU exposed groups and audio emissionsfor vessels containing UCA. Since the ∼50 μm thick vessel wall is surrounded by extra-and intra-luminal buffer it is unlikely that boiling occurs in this model. The detected drivevoltage fluctuations are therefore likely to be due to reflected power from cavitating bubbles.The presence of broadband emission spikes and their approximate temporal coincidence withdrive fluctuations provided further evidence that the detected broadband emissions arose fromacoustic cavitation bubble activity (Neppiras and Coakley 1979, McLaughlan et al 2010).
Audible emission plots only indicated cavitation for 5.0 MPa PNP exposed vessels withor without microbubbles. However the lack of audible emissions for 4.3 MPa ± SonoVuemay not be definitive as both broadband emissions and drive voltage fluctuations suggestedthat acoustic cavitation had occurred. The absence of detected audible emissions may bebecause of the lower sampling frequency used (44 kHz compared to broadband sampling at20 MHz) or it could be due to an inherently lower level of emissions in the lower frequencyrange, which may be of amplitude indistinguishable from noise. Additionally, it is possiblethat the cavitation activity registered, particularly when contrast was not present, occurrednot only inside the vessel but also in the surrounding medium, as the HIFU beam is wider(FWHM 1.9 mm PNP profile) than the vessel diameter (<600 μm). McLaughlan et al (2010)observed that audible emissions caused by boiling in tissue are characterized by frequenciesbelow 10 kHz, whereas in degassed water emissions were centred around 17 kHz. They alsofound that cavitation in water generates discrete spikes rising from a low baseline, whereas intissue there was a continuously raised level with discrete spikes extending above it. Studiesperformed in the absence of a vessel to investigate cavitation in MOPS buffer, with or withoutcontrast agent, showed that for the exposures used in this study, cavitation always occurred(figure 9). Where SonoVue was present the broadband emissions were characterized by peaksof high amplitude which decayed after <0.2 s, suggesting that the microbubbles were rapidlydestroyed. The characteristics of the broadband spikes and audible emissions seen here weresimilar to those observed in degassed water by McLaughlan et al (2010).
Comparison of figures 8(a) and 9 suggests that the acoustic cavitation detected duringvessel exposure is likely to have occurred outside the vessel. However, it is probablethat cavitation also took place in the much smaller volume of fluid within the vessel as
2302 A Tokarczyk et al
increased vessel permeability in vessels containing intraluminal UCA strongly suggests thatit was induced by HIFU exposure. Furthermore microscopic investigation reveals that thehyperintense spots are more likely to occur on the inside upper vessel wall which may be indirect contact with microbubbles. Unfortunately, it is not possible to separate these effects,because the HIFU exposures appear to have been significantly above the acoustic cavitationthreshold, and thus cavitation was likely within much of the focal region. Cavitation is unlikelyto occur outside the vessel in vivo, and therefore it may be useful to investigate methodsfor suppressing this activity in the isolated, cannulated vessel model e.g. by degassing theextraluminal buffer. These findings need to be further explored in future experiments.
In summary, this novel experimental setup allows vital vessel maintenance for atleast 4 h, and selective assessment of endothelial and SMC functionality before and afterultrasound exposure, with simultaneous cavitation monitoring and microscopy imaging. Itallows alignment of the vessel in the focus of the ultrasound beam to within ± 150 μm. Thesepreliminary experiments have shown that the HIFU exposures of vessels (with and withoutUCA) used result in little change in SMC functionality. The response of ECs to Ach wassignificantly reduced in vessels with UCA exposed to 5.0 MPa PNP. Histological stainingshowed abnormal uptake of eosin stain in all exposed vessels except that exposed to 4.3 MPain the absence of intraluminal bubbles.
The system has been demonstrated to be fit for purpose, and comprehensive studies ofHIFU and diagnostic exposures of vessels with and without intraluminal contrast agent arecurrently being undertaken.
Acknowledgments
The authors would like to thank Victoria Bull for her help with cavitation detection, SimonWoodford for the software used to determine the vessel diameters, Chris Bunton and CraigCummings for building the tank and vessel chamber and John Civale and Eleanor Stride forinvaluable discussions. They would also like to acknowledge funding from the UK Engineeringand Physical Sciences Research Council (EP/E029612/1 and EP/F029217/1).
References
Aalkjær C and Nilsson H 2005 Vasomotion: cellular background for the oscillator and for the synchronization ofsmooth muscle cells Br. J. Pharmacol. 144 605–16
Bakker E, Pistea A and Van Bavel E 2008 Transglutaminases in vascular biology: relevance for vascular remodelingand atherosclerosis J. Vasc. Res. 45 271–8
Brayden J, Halpern W and Brann L 1983 Biochemical and mechanical properties of resistance arteries fromnormotensive and hypertensive rats Hypertension 5 17–25
Bull V, Civale J, Rivens I H and ter Haar G R 2011 Cavitation detection using a fibre-optic hydrophone: a pilot studyAIP Conf. Proc. 1359 85–90
Caskey C F, Stieger S M, Qin S, Dayton P A and Ferrara K W 2007 Direct observations of ultrasound microbubblecontrast agent interaction with the microvessel wall J. Acoust. Soc. Am. 122 1191–200
Chen H, Brayman A A, Bailey M R and Matula T J 2010a Blood vessel rupture by cavitation Urol. Res. 38 321–6Chen H, Brayman A A, Kreider W, Bailey M R and Matula T J 2011 Observation of translation and jetting of
ultrasound-activated microbubbles in mesenteric microvessels Ultrasound Med. Biol. 37 2139–48Chen H, Brayman A A, Matula T J and Evan A P 2010b Vascular damage by ultrasound-activated microbubble
induced vessel invagination IEEE Ultrasonics Symp. pp 678–81Chen L, Bouley D, Yuh E, D’arceuil H and Butts K 1999a Study of focused ultrasound tissue damage using MRI and
histology J. Magn. Reson. Imaging 10 146–53Chen L, ter Haar G, Robertson D, Bensted J P M and Hill C R 1999b Histological study of normal and tumor-bearing
liver treated with focused ultrasound Ultrasound Med. Biol. 25 847–56
Ultrasound exposure of isolated blood vessels 2303
Christiansen J P, French B A and Klibanov A L 2003 Targeted tissue transfection with ultrasound destruction ofplasmid-bearing cationic microbubbles Ultrasound Med. Biol. 29 1759–67
Cretel E, Pierres A, Benoliel A-M and Bongrand P 2008 How cells feel their environment: a focus on early dynamicevents Cell. Mol. Bioeng. 1 5–14
Dalecki D 2007 WFUMB Safety Symposium on Echo-Contrast Agents: bioeffects of ultrasound contrast agentsin vivo Ultrasound Med. Biol. 33 205–13
Earley S, Resta T C and Walker B R 2004 Disruption of smooth muscle gap junctions attenuates myogenicvasoconstriction of mesenteric resistance arteries Am. J. Physiol. Heart Circ. Physiol. 287 H2677–86
Furchgott R, Carvalho M, Khan M and Matsunaga K 1987 Evidence for endothelium-dependent vasodilation ofresistance vessels by acetylcholine Blood Vessels 24 145–9
Henderson P W, Lewis G K, Shaikh N, Sohn A, Weinstein A L, Olbricht W L and Spector J A 2010 A portablehigh-intensity focused ultrasound device for noninvasive venous ablation J. Vasc. Surg. 51 707–11
Herrmann K S 1983 Platelet aggregation induced in the hamster cheek pouch by a photochemical process with excitedfluorescein isothiocyanate-dextran Microvasc. Res. 26 238–49
Hitchcock K E et al 2010 Ultrasound-enhanced delivery of targeted echogenic liposomes in a novel ex vivo mouseaorta model J. Control. Release 144 288–95
Huang J, Zhang X and Mcnaughton P A 2006 Modulation of temperature-sensitive TRP channels Semin. Cell Dev.Biol. 17 638–45
Hwa J J, Ghibaudi L, Williams P and Chatterjee M 1994 Comparison of acetylcholine-dependent relaxation in largeand small arteries of rat mesenteric vascular bed Am. J. Physiol. 266 H952–8
Hynynen K, Colucci V, Chung A and Jolesz F 1996 Non-invasive arterial occlusion using MRI-guided focusedultrasound Ultrasound Med. Biol. 22 1071–7
Ichihara M, Sasaki K, Umemura S-I, Kushima M and Okai T 2007 Blood flow occlusion via ultrasound image-guidedhigh intensity focused ultrasound and its effect on tissue perfusion Ultrasound Med. Biol. 33 452–9
Kaneko Y, Maruyama T, Takegami K, Watanabe T, Mitsui H, Hanajiri K, Nagawa H and Matsumoto Y 2005Use of a microbubble agent to increase the effects of high-intensity focused ultrasound on liver tissue Eur.Radiol. 15 1415–20
Kimura M, Jefferis A-M, Watanabe H and Chin-Dusting J 2002 Insulin inhibits acetylcholine responses in rat isolatedmesenteric arteries via a non-nitric oxide nonprostanoid pathway Hypertension 39 35–40
Leskinen J J and Hynynen K 2012 Study of factors affecting the magnitude and nature of ultrasound exposure within vitro set-ups Ultrasound Med. Biol. 38 777–94
Looft-Wilson R C and Gisolfi C V 2000 Rat small mesenteric artery function after hindlimb suspension J. Appl.Physiol. 88 1199–206
Luo W, Zhou X, Ren X, Zheng M, Zhang J and He G 2007 Enhancing effects of SonoVue, a microbubble sonographiccontrast agent, on high-intensity focused ultrasound ablation in rabbit livers in vivo J. Ultrasound Med.26 469–76
Martin E M, Duck F A and Winlove C P 2012 Thermally-mediated ultrasound-induced contraction of equine musculararteries in vitro and an investigation of the associated cellular mechanisms Ultrasound Med. Biol. 38 152–61
McIntyre C A, Williams B C, Lindsay R M, Mcknight J A and Hadoke P W F 1998 Preservation of vascularfunction in rat mesenteric resistance arteries following cold storage, studied by small vessel myography Br.J. Pharmacol. 123 1555–60
Mclaughlan J, Rivens I, Leighton T and ter Haar G 2010 A study of bubble activity generated in ex vivo tissue byhigh intensity focused ultrasound Ultrasound Med. Biol. 36 1327–44
Mehta D and Malik A B 2006 Signaling mechanisms regulating endothelial permeability Physiol. Rev. 86 279–367Miller D L 2007a Overview of experimental studies of biological effects of medical ultrasound caused by gas body
activation and inertial cavitation Prog. Biophys. Mol. Biol. 93 314–30Miller D L 2007b WFUMB safety symposium on echo-contrast agents: in vitro bioeffects Ultrasound Med.
Biol. 33 197–204Miller D L and Thomas R M 1995 Ultrasound contrast agents nucleate inertial cavitation in vitro Ultrasound Med.
Biol. 21 1059–65Morris P, Hurrell A, Shaw A, Zhang E and Beard P 2009 A Fabry–Perot fiber-optic ultrasonic hydrophone for the
simultaneous measurement of temperature and acoustic pressure J. Acoust. Soc. Am. 125 3611–22Mulvana H, Stride E, Hajnal J V and Eckersley R J 2010 Temperature dependent behaviour of ultrasound contrast
agents Ultrasound Med. Biol. 36 925–34Nakamura Y and Wayland H 1975 Macromolecular transport in the cat mesentery Microvasc. Res. 9 1–21Neppiras E A and Coakley W T 1979 Acoustical cavitation in a focused field in water at 1 MHz J. Sound Vib.
45 341–73Nilsson H and Aalkjaer C 2003 Vasomotion: mechanisms and physiological importance Mol. Interv. 3 79–89, 51Nishimura J, Khalil R and Breemen C V 1989 Agonist-induced vascular tone Hypertension 13 835–44
Pistea A, Bakker E, Spaan J and Van Bavel E 2005 Flow inhibits inward remodeling in cannulated porcine smallcoronary arteries Am. J. Physiol. Heart Circ. Physiol. 289 H2632–40
Rivens I H, Rowland I J, Denbow M, Fisk N M, ter Haar G and Leach M O 1999 Vascular occlusion using focusedultrasound surgery for use in fetal medicine Eur. J. Ultrasound 9 89–97
Rowland I J, Rivens I H, Chen L, Lebozer C H, Collins D J, ter Haar G and Leach M O 1997 MRI study of hepatictumours following high intensity focused ultrasound surgery Br. J. Radiol. 70 144–53
Samuel S, Cooper M A, Bull J L, Fowlkes J B and Miller D L 2009 An ex vivo study of the correlation betweenacoustic emission and microvascular damage Ultrasound Med. Biol. 35 1574–86
Schneider M, Broillet A, Tardy I, Pochon S, Bussat P, Bettinger T, Helbert A, Costa M and Tranquart F 2012 Useof intravital microscopy to study the microvascular behaviour of microbubble-based ultrasound contrast agentsMicrocirculation 19 245–59
Scotland R S, Chauhan S, Davis C, Felipe C D, Hunt S, Kabir J, Kotsonis P, Oh U and Ahluwalia A 2004 Vanilloidreceptor TRPV1, sensory C-fibers, and vascular autoregulation. A novel mechanism involved in myogenicconstriction Circ. Res. 95 1027–34
Smith R A and Bacon D R 1990 A multiple-frequency hydrophone calibration technique J. Acoust. Soc.Am. 87 2231–43
Stieger S M, Caskey C F, Adamson R H, Qin S, Curry F-R E, Wisner E R and Ferrara K W 2007 Enhancement ofvascular permeability with low-frequency contrast-enhanced ultrasound in the chorioallantoic membrane modelRadiology 243 112–21
Sun D, Messina J, Kaley G and Koller A 1992 Characteristics and origin of myogenic response in isolated mesentericarterioles Am. J. Physiol. 263 H1486–91
ter Haar G 2009 Safety and bio-effects of ultrasound contrast agents Med. Biol. Eng. Comput. 47 893–900Unger E C, Matsunaga T O, Mccreery T, Schumann P, Sweitzer R and Quigley R 2002 Therapeutic applications of
microbubbles Eur. J. Radiol. 42 160–8Van Bavel E, Giezeman M J M M, Mooij T and Spaan J A E 1991 Influence of pressure alternations on tone and
vasomotion of isolated mesenteric small arteries of the rat J. Physiol. 436 371–83Van Bavel E, Wesselman J P M and Spaan J A E 1998 Myogenic activation and calcium sensitivity of cannulated rat
mesenteric small arteries Circ. Res. 82 210–20Van Haaren M A P, Van Bavel E, Vink H and Spaan J A E 2003 Localization of the permeability barrier to solutes in
isolated arteries by confocal microscopy Am. J. Physiol. Heart Circ. Physiol. 285 H2848–56Van Wamel A, Kooiman K, Harteveld M, Emmer M, Cate F J T, Versluis M and Jong N D 2006 Vibrating microbubbles
poking individual cells: drug transfer into cells via sonoporation J. Control. Release 112 149–55Yuan S Y and Rigor R R 2010 The endothelial barrier Regulation of Endothelial Barrier Function ed D N Granger
and J P Granger (San Rafael, CA: Morgan & Claypool Life Sciences)Zderic V, Brayman A A, Sharar S R, Crum L A and Vaezy S 2006 Microbubble-enhanced hemorrhage control using
high intensity focused ultrasound Ultrasonics 45 113–20
