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Intravascular mural thrombi produced by acoustic microstreaming
Ultrasound Med. Biol., Vol. 3, pp. 191-203. Pergamon Press, 1977. Printed in Great Britain
INTRAVASCULAR MURAL THROMBI PRODUCED BY ACOUSTIC MICROSTREAMING* A. R. WILLIAMS Department of Medical Biophysics, University of Manchester, Manchester, England. (First received 6 August 1976; and in final form 31 March 1977) Abstraet--A discrete portion of the wall of an intact blood vessel may be driven to oscillate at an ultrasonic frequency in vivo by the external application of a vibrating metal probe. The hydrodynamic forces generated within the local intravascular microstreaming field at the site of contact with the probe tip increase with increasing amplitude of oscillation of the ultrasonic driver. Under the appropriate experimental conditions one may produce (1) no detectable effects; (2) adhesion of platelets to apparently intact endothelium; (3) fibrin-free mural aggregates of platelets with the concomitant production of platelet micro-emboli; (4) mural thrombi of platelets permeated with fibrin and enveloped within a mixed clot and (5) obvious damage to the vessel wall and its endothelium with widespread clot formation. This technique might be of use in the in vivo screening of drugs which may interact with the haemostatic system. Key words: Acoustics, Ultrasonics, Ultrasonic damage, Platelet damage, Endothelial damage, Blood clot formation, Intravascular microstreaming. INTRODUCTION
Significant technical problems are associated with the production of discrete wellcharacterized thrombi at specific sites within the intact vascular tree of an experimental animal in vivo. If these thrombi are to be of use in the evaluation of the in vivo effectiveness of new pharmacologically active agents, which might promote or interfere with the haemostatic system, they have to be produced in a highly reproducible manner under carefully controlled experimental conditions. Numerous experimental systems have been proposed to produce adequate mural thrombi in vivo (Henry, 1962). These techniques are in general divisible into two main groups; one group damages and/or removes the endothelium (e.g. by pinching a vessel with fine forceps; electrical coagulation of a portion of a vessel wall (Spilker et al., 1973) or by the insertion of a rigid probe or inflatable balloon to scour the vessel wall (Palko et al., 1964; Baumgartner et al., 1971)) while the other group modifies the chemical environment adjacent to the endothelium so that platelets are attracted to it and adhere (e.g. by the electrodynamic diffusion of platelet aggregating agents through the vessel wall, (Born, I973) or the thermal precipitation of erythrocyte contents by means of a finely *Work accomplished under the partial support of contract number FDA 3177-75 (J) from the Bureau of Radiological Health, United States Food and Drug Administration.
focussed laser beam (Poliwoda et al., 1973; Wiedeman, 1973)). Platelets contribute to clot formation in the above experimental models by first adhering to "altered" endothelium or exposed subendothelial structures and subsequently by participating in and accelerating the conversion of fibrinogen to fibrin (Thomas, 1972; Vermylen et al., 1973). However, platelets may not have to adhere to a "damaged" endothelial surface before they are stimulated to participate in clot formation (Schiffman et al., 1973). Human platelets are extremely susceptible to damage by hydrodynamic shear stresses and undergo membrane rupture and the loss of intracellular components when exposed to stresses greater than 50 to 100 dyn cm -2 for 5 min (Brown et al., 1975). Similarly, Williams (1974) has demonstrated rupture of the platelet plasma membrane and the release of incorporated serotonin after millisecond exposures to shear stress within the acoustic microstreaming field generated around a steel wire oscillating transversely at 20kHz. Rupture of the plasma membrane increased platelet "stickiness" (i.e. that tendency of platelets to adhere to adjacent surfaces and to participate in spontaneous aggregation) (O'Brien, 1969) and increased the availability of platelet procoagulant agents (PF-3) which subsequently accelerated the rate of fibrin formation in vitro (Glover et al., 1975; Williams et al., 1976, 1977). Thus platelets which have suffered mem191
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brane damage following exposure to hydrodynamic shear stress are more adhesive than their controls and may be already "primed" to participate in fibrin formation. This paper describes a novel technique whereby platelets within the fluid blood are damaged by hydrodynamic streaming forces while adjacent to the vascular endothelium so that they might themselves initiate formation of a discrete mural thrombus in vivo without recourse to prior "alteration" or damage to the vascular endothelium.
MATERIALS AND METHODS
The ultrasonic driver consists of a short resonant length (1.69cm) of ll5/~m radius steel wire clamped at one end to the tip of a stainless steel velocity transformer. This velocity transformer was bonded with epoxy resin to a PZT 4 ultrasonic transducer and driven to oscillate in its longitudinal mode at 20 kHz. The wire was clamped at right angles to the long axis of the transducer/velocity transformer so that a flexural (transverse) standing wave was set up along the length of the wire (Williams et al., 1970). Thus, portions of the wire were driven to oscillate transversely at 20kHz in a plane between the limits indicated diagrammatically by the broken lines in Fig. 1. The amplitude of transverse oscillation of the wire increased as the power to the ultrasonic transducer was increased. At all times, the maximum value of transverse oscillation was found at the hemispherical tip of the free end of the wire; all displacement amplitude values quoted in this article (up to the maximum of about 30/xm) refer to the displacement amplitude measured at this free end of the wire in its unloaded condition (see Discussion). The transducer assembly was firmly mounted in rubber and held so that the tip of the wire was positioned at the focus of a low power (up to x30) steroscopic dissecting microscope. When the hemispherical tip of the oscillating wire is in contact with a fluid medium it acts as a local generator of small scale (d.c.) acoustic microstreaming (Williams et al., 1970; Williams, 1974). This localized streaming field is seen as symmetrical eddy currents of fluid immediately adjacent to the wire tip. Surface waves (ripples) were visible when the liquid meniscus met the wire at any point other than at a displacement node. These ripples were also seen in the thin film
P iii
I
iI x
w) i
Fig. 1. A diagrammatic representation of the sonication system. A resonant length of steel wire (W) was clamped at right angles to the tip of a steel probe (P) oscillating in its longitudinal mode at 20kHz. This configuration produced a flexural (transverse) standing wave pattern along the length of the wire as indicated by the broken lines, which represent the greatly exaggerated limits of displacement amplitude. The hemispherical tip of the free end of the wire was pressed against the outside wall of an exposed but intact blood vessel in vivo and driven so that a portion of the vessel wall was made to oscillate with the wire tip.
of saline bathing the exposed blood vessels and were used to "tune" the transducer assembly so that the wire was always oscillating with maximum displacement amplitude for that particular value of applied electrical power. Young adult male mice (Manchester strain of albino Swiss) were anaesthetized with Nembutal (Pentobarbital) at a dosage of 60mg/kg administered intraperitoneally. A flap of skin over the inside of the thigh was carefully resected to expose the femoral artery and vein, and the transparent thin fibrous sheet (the fascia lata) which overlies the vessels was not disturbed. The mouse was placed on a thermostated microscope stage which was raised until the fascia lata over the chosen blood vessel pressed against the wire tip (Fig. 1). The terminal portion of the leg under investigation was attached to a micromanipulator to damp out spontaneous movements of the limb and enable minute adjustments to be made in the height and position of the blood vessel relative to the wire tip (which remained at the focus of the dissecting microscope). The height of the limb was adjusted until the wire tip made a visible indentation in the vessel wall (i.e. it protruded between about 20 and 50% of the vessel diameter). This indentation was approximately conical in shape and resembled the indentation made by
Intravascular mural thrombi produced by acoustic microstreaming pushing a finger into an air-filled balloon. The hemispherical wire tip was the only portion of the wire physically in contact with the vessel wall. This wall was held in intimate contact with the wire tip by a small elastic restoring force from the deformed membrane and by the intraluminal pressure generated within the intact vessel by the heart of the animal (i.e. by its own blood pressure). This pressure ensured good coupling so that the portion of the vessel wall in contact with the wire tip was also driven to oscillate at 20 kHz. The oscillating portion of the vessel wall then acted as a local generator of acoustic microstreaming within the fluid blood (Fig. 2). The exposed blood vessels were kept moist by the repeated application of small quantities of isotonic saline at a temperature of 38°C. At the end of the sonication procedure, a small drop of an inert colouring material (India ink) was allowed to run down the wire to mark the site of irradiation. Once dry, the mark remained as a discrete spot despite repeated washings with various fixatives and staining reagents, and did not appear to alter the morphology of the underlying tissues. After irradiation, the blood of the animal was allowed to flow through the exposed vessels for various times within the range 0 to 3000 sec before they were tied off with surgical sutures and fixed or frozen in situ. Vessels which had been tied off with surgical sutures and partially fixed in situ with isotonic aldehydes were excised, fixed, dehydrated and embedded in paraffin wax. Alternatively, vessels were tied off and
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frozen in situ with isopentane which had been cooled in liquid nitrogen, excized, mounted in liver on a metal block and sectioned in a Slee cryostat. Thin sections were stained with Haematoxylin/Eosin, Masson's Trichrome or M.S.B. (which stains fibrin red and platelets blue). The freezing and thawing associated with obtaining frozen sections, combined with the low osmotic strength of the first stage of the staining procedure for Masson's trichrome (aqueous Haematoxylin), lysed the erythrocyte population. Samples destined for electron microscopy were tied off with surgical sutures and bathed in isotonic cacodylate buffer pH 7.4 containing 2% Glutaraldehyde for 15 min. The rigid vessels were excized, fully fixed in the same fixative for 1 hr, post fixed in osmium tetroxide, dehydrated and embedded in e p o x y resin (AralditeR). Thin sections were stained with uranyl acetate and lead nitrate and examined in a Phillips EM201 electron microscope. RESULTS
A separate series of experiments were undertaken to investigate the effect of contact pressure between the wire tip and the vessel wall on the results presented below. An adult male mouse was anaethetized and a portion of its small intestine with its intact mesenteric circulation was placed on a heated (38°C) microscope stage. A 300 to 350/xm diameter blood vessel was transilluminated and the transversely oscillating wire (mounted on a micromanipulator) was advanced until it made contact with the vessel wall. The wire was driven at a maximum tip displace-
Fig. 2. This diagram shows the relative dimensions of the structures at the site of contact with the wire (W)tip. The vessel wall (V) was displaced by a static contact force a distance, d, which was maintained between 20 and 50% of the vessel diameter (D). When the vessel wall was driven to oscillate, symmetrical eddies of acoustic microstreaming could be seen within the intact blood vessel, as indicated by the broken lines.
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ment amplitude of 8 or 12/~m. The depth of penetration of the wire tip into the intact blood vessel (expressed as a percentage of the vessel diameter) was taken as an indirect estimate of the contact pressure since a more direct measurement was not available. Under the appropriate illumination conditions, intravascular acoustic microstreaming is seen as two symmetrical dark regions approximately egg-shaped situated on either side of the mid point of contact with the wire tip. The diameter of these "shadows" increases rapidly with increasing penetration of the wire over the range 0 to about 10 or 15% of the vessel diameter; the wire displacement amplitude being held constant. Further penetration of the wire up to about 60% of the vessel diameter had no visible effect on the microstreaming patterns (Fig. 2). Deeper penetrations were not attempted. The histological results reported below were obtained from mouse femoral arteries and veins where the intravascular microstreaming eddies could not be seen. Consequently, histological sections were cut from femoral arteries and veins from different legs of the same mouse after they had been irradiated at the same wire displacement amplitude (8 or 12/~m) for the same time (5 min) but at 20 or 50% penetration of the vessel diameter. The results obtained were sufficiently similar to indicate that the extent of wire penetration within this range had no detectable effect on the histological appearance of the vessel. There was also no sign of thermal damage at the site of contact with the wire tip. Light and electron microscopical examination of the vessel wall at the site of contact with the wire tip showed transverse wire displacement amplitudes of the order of 2 to 4 p~m at 20 kHz produced no damage to the vascular endothelium which could not have been caused by the mechanical trauma associated with freezing, cutting, staining or washing the thin sections. However, some histological changes were observed at wire displacement amplitudes greater than 5/~m. In general, the nature and extent of the observed changes was influenced by the displacement amplitude of the wire, the duration of the ultrasonic exposure and the time interval between exposure and fixation in situ prior to removal for histological examination. The role of the vascular endothelium in thrombus formation following ultrasonic
exposure was investigated by means of the electron microscope. Preliminary results showed that occasional platelets (PI) had fused with the endothelium of mouse femoral veins at the site of contact with the wire tip after it had been driven at displacement amplitudes of the order of 5 to 8 ~m for 5 min (Fig. 3). These fused platelets had undergone the release reaction (i.e. were degranulated), contained bundles of microtubules (arrowed) which extended into projections from the platelet surface and frequently exhibited large-scale rupture of the plasma membrane with loss of cytoplasmic material. Each fused platelet had one or more intact or damaged platelets (P2) adhering to it. Apart from these occasional fused platelets (approximately one for each 50 endothelial cells viewed as thin sections cut in a plane at right angles to the long axis of the vessel) there was no obvious damage to the endothelium, which remained apparently intact and full of pinocytotic vesicles (Fig. 3). Light micrographic investigations of frozen and wax embedded blood vessels which had been sonicated at wire displacement amplitudes in the range 10 to 15 ~m showed the presence of mural thrombi at the site of contact with the wire tip. The size and type of mural thrombus varied with the amplitude of oscillation of the ultrasonic driver and the time interval between irradiation and fixation. Mouse femoral veins which had been subjected to a wire displacement amplitude of about 10/.~m for 5 min and then left for a further 20 min with blood flowing over the irradiated area produced large "loose" mural aggregates of platelets (Fig. 4). These large aggregates could achieve a diameter of up to 05 mm and a length of several millimetres. Histologically, these mural thrombi consisted of a core of densely packed (but not fused) platelets surrounded by a zone of loosely adhered platelets, the whole being infiltrated with leucocytes. There was no visible evidence of fibrin formation or erythrocyte participation (Fig. 4). Figure 5 is light photomicrograph of a longitudinal cryostat (frozen) section of a mouse femoral vein stained by the trichrome procedure (which has lysed the erythrocyte population). The oscillating wire was positioned upstream of the field of view and driven at a displacement amplitude of 12 ~m for 20 min. A large "loose" mural aggregate of platelets similar to that depicted in Fig. 4 was sub-
Intravascular mural thrombi produced by acoustic microstreaming
Fig. 3. This electron micrograph shows the intact and apparently undamaged endothelium (E) of a mouse femoral vein which had been subjected to a wire displacement amplitude of about 8/xm for 5 min. The platelet Pm (which has suffered extensive rupture and removal of its plasma membrane and the loss of some intracellular organelles) has attached itself to or fused with the endothelium where it has attracted another platelet (P2) which has adhered to it forming a micro-mural aggregate. The arrow indicates the position of a bundle of microtubules. (1/~m = 2.64 cm.)
Fig. 4. Light micrograph of a transverse cryostat (frozen) section through a mouse femoral vein (V) which had been irradiated at 10 tzm for 5 min and blood left to flow through the vessel for a further 20 min before fixation and removal. The lumen of the vessel was largely occluded by a "loose" mural aggregate of fibrin-free platelets (P) infiltrated with leucocytes. Erythrocytes were lysed during the staining procedure with Masson's trichrome. (100 ~m = 1.25 cm.)
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Fig. 5. Longitudinal cryostat section through a mouse femoral vein stained with Masson's trichrome. The wire was placed upstream of the field of view and driven at an amplitude of about 12 p.m for 20 min where it produced a "loose" mural aggregate of platelets similar to that seen in Fig. 3. The vessel was frozen with cold isopentane while the wire was still oscillating. Numerous fibrin-free micro-emboli of platelets (A) can be seen en r o u t e to the heart and lungs through the valve (V). The arrow indicates the direction of blood flow. (100 ~m = 2.18 cm.)
Fig. 6. A transverse section through the femoral artery (A) and vein (V) of a mouse after the wire had been applied to the fascia lata overlying the inflated vein (marked with India ink, I) and driven at an amplitude of about 20/zm for 5 rain. A compact mural aggregate of platelets (T) is seen within a mixed clot on that portion of the wall which, when inflated, was closest to the wire tip. The vessels were fixed in situ, embedded in paraffin wax and thin sections stained with Haematoxylin/Eosin. (100 krm = 1.2 cm.)
Intravascular mural thrombi produced by acoustic microstreaming
Fig. 7. Another section at a higher magnification of the same mural thrombus presented in Fig. 6, but stained by the M.S.B. procedure to demonstrate fibrin. A dense band of fibrin (F) is visible at the endothelial junction from which fibrin strands permeate the platelet aggregate (A). The arrow indicates a mixed thrombus consisting predominantly of erythrocytes enmeshed in a fibrin network. (40/~m = 1.88 cm.)
Fig. 8. A longitudinal cryostat section through a mouse femoral vein (V) which had been completely thrombosed following 5 min irradiation at a wire displacement amplitude of about 30 ~m. The wire was placed upstream of the field of view where it had damaged the vascular endothelium. The lumen of the vessel contains a densely staining fibrous mass (F) consisting mainly of fibrin embedded in a cytoplasmic material which may once have been platelets, and a homogeneous background material (B) which resembles a typical fresh mixed clot after the erythrocytes have been lysed by the staining procedure. (100 tim = 1.84 cm.)
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Intravascular mural thrombi produced by acoustic microstreaming
sequently found at the site of contact with the wire tip. However, in this case the vessel was not tied off and fixed in situ but merely clamped shut with forceps while the wire was still oscillating. Isopentane, cooled in liquid nitrogen, was poured on to the exposed vessel as soon as it was clamped; this rapidly froze the blood and maintained the correct spatial relationship of the intravascular contents. Numerous small "loose" platelet aggregates (A) can be seen floating freely within the lumen of the vessel, frequently in association with leucocytes (Fig. 5). These micro-aggregates are visible on both sides of the venous valves (V) and are presumably being borne by the circulation to the heart and lungs. Mural thrombi produced near the wire tip at higher displacement amplitudes (e.g. 15 to 20 tzm) were more compact than those described above, and were frequently found in association with erythrocytes. Figure 6 is a low power light micrograph of a section through the femoral artery (A) and femoral vein (V) of a young adult male mouse, stained with Haematoxylin/Eosin. India ink (I) marks the site at which the wire tip was applied to the fibrous fascia lata which was in contact with the femoral vein when it was fully inflated with blood. The thin muscular coat of the vein was not enough to maintain its circular cross-section when the vessel was cut and embedded in paraffin wax. The wire was driven at a displacement amplitude of about 20 txm for 5 min, and blood was allowed to flow over the irradiated region for 20min before the vessel was tied off and fixed in situ with formal saline. Most of the free blood cells (i.e. those cells not attached to the vessel walls or participating in thrombus formation) were lost during the fixation, embedding, sectioning and staining procedures. A mural thrombus (T) consisting of tightlypacked platelets was visible on that portion of the wall immediately adjacent to the site of contact with the wire tip (Fig. 6). Serial sections showed that this thrombus was at least 3 mm long. The platelet thrombus was enclosed within a mixed thrombus consisting mainly of erythrocytes. Another mixed thrombus was found on the opposite wall of the same vessel, i.e. the wall which was furthest away from the site of contact with the wire tip, but this thrombus did not contain a region consisting predominantly of platelets.
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Figure 7 is a light micrograph of another section of the same mural thrombus of tightly packed platelets presented in Fig. 6, but taken at a higher magnification. This section was stained by the M.S.B. procedure which specifically stains fibrin bright red while platelets appear blue. A dense red band was visible at the platelet/endothelium junction which merged into the remainder of the platelet mass which was lilac/purple in colour and permeated with thin lines of red. Strands of fibrin could also be seen amongst the erythrocytes at the periphery of the platelet thrombus. Blood vessels irradiated at wire displacement amplitudes greater than about 20 ~m showed immediate signs of damage. Blood was observed to seep from the vessels into the isotonic saline bathing fluid. Gross histological damage was observed in both the muscular walls of the irradiated vessels and the vascular endothelium. The flow of blood within the vessels was grossly perturbed by the formation of two large rapidly-rotating vortices which completely filled the lumen of the vessel. Femoral veins having a diameter of up to 2 mm could be completely occluded by thrombus formation within a few minutes of irradiation at a wire displacement amplitude of the order of 30 tzm. Figure 8 is a representive light micrograph of a cryostat (frozen) section stained with Masson's trichrome of a mouse femoral vein (V) which had been completely occluded by a thrombus. The wire was placed upstream of the field of view and driven at a displacement amplitude of about 30/zm for 5 min. The direction of blood flow was determined from the orientation of the vessel on the metal mounting block in the cryostat and the appearance of valves within the veins. The vessel contents are clearly divisible into two components: one is the homogeneous background material (B) which consists of erythrocyte ghosts (lysed by the staining procedure) and platelets bound together with fibrin and the other is a dense fibrous material (F) which appears to consist mainly of fibrin and a cellular material which may once have been platelets (Fig. 8). DISCUSSION
Symmetrical intravascular eddy currents may be seen within the blood adjacent to the tip of a transversely oscillating wire after it has been applied to the outside of an intact
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mesenteric blood vessel in vivo. This intravascular streaming appears to be identical with the acoustic microstreaming field which is generated when the same oscillating wire is immersed in a homogeneous fluid (Williams et al., 1970). This indicates that a discrete portion of the blood vessel has been driven to oscillate at an ultrasonic frequency (20 kHz) so that it acts as a local generator of smallscale acoustic microstreaming within the fluid blood. The only practicable measurement of the contact force between the wire and the inflated blood vessel was the depth of penetration of the wire tip (expressed as a percentage of the vessel diameter). It has been determined experimentally that both the visual appearance of the microstreaming eddies and their histological sequelae were virtually identical whether the penetration depth was 20 or 50% of the vessel diameter. These observations, together with the absence of detectable thermal damage to the superficial vascular structures in contact with the wire tip, indicate that the wire was efficiently coupled to the vessel wall. The displacement amplitude of the wire tip could not be measured during the course of the experiment as it was buried within the lumen of the vessel. The frequent occurrence of slow spontaneous limb movements meant that the operator was fully employed maintaining the correct depth of wire penetration and ensuring that the wire was oscillating with maximum efficiency for that value of applied electrical power (as indicated by surface waves on the saline bathing the contact site). The displacement amplitudes quoted below were measured at the tip of the wire when the same driving voltage was applied to the piezoelectric transducer, but with the wire in an unloaded condition (i.e. the wire tip was not in contact with the vessel wall). There was no significant change in the displacement amplitude measured at the antinode adjacent to the wire tip when that tip was loaded by contact with an inflated blood vessel. Similar indirect measurements suggest that the increased damping resulting from contact with the vessel wall decreased the displacement amplitude of the tip by less than 10%. Thus, the displacement amplitude values quoted in the text represent the maximum limits of oscillatory motion which could only be obtained within the vessel wall under
conditions of zero damping and 100% efficient coupling. The biological results obtained at each discrete value of transverse wire displacement amplitude are influenced by the duration of the ultrasonic exposure and the time interval between exposure and removal of the vessel for histological examination. Nevertheless, it is convenient to partition the results obtained into five overlapping compartments or groups on the basis of increasing wire displacement amplitude (since this is a measure of this instantaneous hydrodynamic shear stress generated at the blood/endothelial cell interface). Briefly, the groups are: (I) no detectable effects; (2) occasional platelet adhesion to apparently normal endothelium; (3) "loose" mural aggregates of fibrin-free platelets with the concomitant production of platelet microemboli; (4) dense mural thrombi of platelets containing fibrin and associated with mixed blood clots and (5) gross damage to the vessel wall and widespread clot formation which occludes even major blood vessels. Group 1 includes those results obtained at wire displacement amplitudes below about 4t~m at 20kHz where no perturbation of blood flow was observed and histological sections were indistinguishable from their controls. Surface waves were clearly visible on the thin film of saline bathing the outside surface of the vessel during irradiation showing that wire was oscillating. This implies either that the intravascular streaming forces produced at these low wire amplitudes were not large enough to perturb the normal blood flow and damage cellular elements, or that the oscillatory energy was dissipated as "slip" or by the deformation of elastic structures within the vessel wall. Group 2 includes those results obtained between wire displacement amplitudes of 5 and about 8 ~m after 5 min irradiation at 20 kHz. These samples appeared normal with the light microscope but preliminary electron micrographic examination of the site of contact with the wire tip showed that occasional platelets had adhered or fused with what appeared to be normal endothelial surface (Fig. 3). Many of the fused platelets (P1) showed evidence of large-scale membrane rupture and the loss of cytoplasmic structures including secretory granules, mitochondria and dense bodies. Each fused platelet had one or more other platelets (P2) adhering to it forming a micro-mural thrombus (Fig. 3). It
Intravascular mural thrombi produced by acoustic microstreaming
was not possible to determine from the electron micrographs whether the platelet plasma membrane was ruptured before or after it became adhered to the endothelial surface. Two conflicting hypotheses may be proposed to explain the mechanism of this crucial interaction between platelets and endothelium. One is that the intravascular acoustic microstreaming field produced many minute lesions at discrete sites on the endothelial surface. Normal platelets would then be attracted to these sites where they would undergo the release reaction and adhere. If it is assumed that the plasma membrane of each endothelial cell is of uniform susceptibility to ultrasound, then the hydrodynamic streaming forces generated by the microstreaming field ought to produce a uniform region of endothelial cell damage over the entire area adjacent to the wire tip (4.3 × 104/zm2) and not merely at discrete sites. However nonuniformity of the membrane could lead to localized damage at susceptible sites. An alternative hypothesis assumes that the platelet plasma membrane was ruptured before it became adhered to the endothelium. There is no precedent for presuming the spontaneous rupture of the platelet plasma membrane immediately following its adhesion to the endothelium, and yet damaged platelets have been found attached to the endothelial cells immediately after irradiation, suggesting that they were damaged before they became attached. Human platelets are notoriously susceptible to damage by hydrodynamic shear stress (see Introduction) and undergo large scale rupture of the plasma membrane when subjected to the acoustic microstreaming field generated around a steel wire oscillating transversely at 20 kHz (Williams, 1974). The electron microscopic appearance of human and canine platelets which have been partially disrupted in this way closely resemble the mouse platelets PI and P2 depicted in Fig. 3, i.e. most of the intracellular contents remain bound together even though a portion of the plasma membrane has been removed (Williams et al., 1977). The intracellular contents of mouse ascites cells also remain firmly adhered together when portions of the plasma membrane are torn away by the same acoustic microstreaming field (Williams, 1972). It is, therefore, not unreasonable to propose that the same adhesive forces which bind the intracellular components together in mouse platelets might
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(when exposed following membrane rupture) also adhere that same platelet remnant to (undamaged) endothelium. The obvious "stickiness" of ultrasonically generated platelet emboli supports this proposal, as does the absence of re-growth of a mural aggregate following macro-embolization, when the wire has ceased to oscillate. It is of interest that this threshold for mouse platelet damage in vivo (5 to 8/~m) agrees well with the minimum value of about 6 ~m for the release of incorporated serotonin from human platelets irradiated in vitro with the same transversely oscillating wire assembly (Williams, 1974). This confirms the extreme shear sensitivity of platelets since these values correspond to hydrodynamic shear stresses of the order of 100 to 250 dyn cm -2. This result also indicated that at these displacement amplitudes the damping effect of the vessel wall on the oscillatory motion of the wire is negligible and that coupling losses can be ignored. Group 3 results were obtained at wire displacement amplitudes between about 10 and 15/zm. Large mural thrombi of platelets infiltrated with leucocytes were usually found at the site of contact with the wire tip (Fig. 4). These mural aggregates invariably had a "loose" appearance in histological sections with no visible evidence of fibrin formation or erythrocyte involvement. Each mural aggregate "shed", or initiated the local formation of micro-aggregates of fibrin-free platelets which were embolized downstream both during and after the period of irradiation. A sample of this "snowflake-like" stream of platelet micro-emboli is presented in Fig. 5. The growing platelet thrombus occasionally became dislodged from the vessel wall and embolized downstream. Frequently, there was no visible re-growth of a mural thrombus at that site in the absence of the ultrasonic field. Group 3 results appear to be an extension of Group 2 results (i.e. adhesion of platelets to the endothelium) with the additional selfperpetuating cycle of platelet attraction, release, adhesion to the growing aggregate and the attraction of more platelets. This rapid growth without detectable fibrin formation explains the large size and "loose" consistency of Group 3 thrombi and their tendencies to shed or initiate micro-emboli and themselves become dislodged as macroemboli. The frequent absence of re-growth (in the absence of the acoustic microstream-
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ing field) indicates that the endothelium at the site of contact with the wire tip has suffered minimal damage. Group 4 results were obtained at wire displacement amplitudes between about 15 and 20/zm. The mural aggregates of platelets were invariably smaller and more compact than those of Group 3 and were usually surrounded by a mixed thrombus consisting predominantly of erythrocytes (Figs 6 and 7). Mixed thrombi without a central "core" of platelets were frequently found attached to other portions of the vessel wall. Figure 7 is another section of the same mural thrombus presented in Fig. 6 but stained by the M.S.B. procedure to demonstrate the presence of fibrin. The tightly-packed platelet aggregate was seen to be permeated with fibrin strands which appeared to have grown out of the dense band of fibrin which was present at the thrombus-endothelial junction. These fibrin strands were also visible between the erythrocytes enveloping the mural thrombus of platelets (Fig. 7). Group 4 results appear to be similar to those of Group 3 with the exception that there has been the rapid and extensive production of fibrin. The platelet/fibrin mural thrombus has contracted and mixed clot formation has been initiated at its surface. Any portion of the vessel where temporary blood stasis could occur was usually filled with a mixed thrombus which attached itself to the vessel wall (the region diametrically opposite the point of application of the wire tip was one such region). Group 5 results were obtained at wire displacement amplitudes greater than about 20 mm and represent a more extreme version of the results presented in Group 4. The vessel wall and its endothelium were obviously damaged by this large oscillatory motion of the wire and so the effects of the normal haemostatic mechanism of the animal were superimposed upon the effects produced by the acoustic microstreaming field. Complementary information was obtained from visual observation of thrombus induction and sealing within mouse mesenteric vessels which were transilluminated on a heated microscope stage. Mural aggregates grew very rapidly (in less than a second) at these large wire displacement amplitudes and could frequently be seen issuing as a continuous fluid stream of platelets from the site of irradiation. This flexible gelatinous mass of
aggregated (and partially disrupted) platelets is extremely "sticky" and adhered itself to the endothelial surface at every contact. Simultaneously, fibrin formation is occurring within the platelet aggregate rendering it progressively more rigid and securing its attachment to the vessel wall. The fibrous material (F) in Fig. 8 appears to be the solidified remains of the original fluid platelet thrombus (now completely permeated with fibrin) which has become attached at all points around the vessel circumference. The blood stasis induced by this solidifying "plug" enabled fibrin formation to occur within the fluid blood giving the mixed clot seen as the homogeneous background material (B) in Fig. 8. Thus, different facets of the haemostatic mechanism as it operates in vivo may be selectively produced at adjacent sites within the same experimental animal merely by the choice of the appropriate wire displacement amplitude. For any given animal anaesthetized by the method of choice, it should be possible to determine the optimum combination of amplitude, frequency of oscillation and duration of exposure which is required to produce no detectable effect (Group 1), platelet adhesion without apparent aggregation (Group 2), mural aggregates without detectable fibrin formation (Group 3), mural aggregates with fibrin formation (Group 4) and, finally, gross damage to the vessel wall and its endothelium with widespread thrombus formation (Group 5). This technique ought, therefore, to be of use in the in vivo screening of pharmacologically active agents which might enhance or interfere with various aspects of the complex haemostatic system. Acknowledgements--I wish to thank Mr John Sherwin for his invaluable technical assistance, Mrs W. Staley and Mr R. Ellis for the photography, Miss N. Osborn for the artwork and Miss K. Holt for typing the manuscript. REFERENCES Baumgartner, H. R., Stemerman, M. B. and Spaet, T. H. (1971) Adhesion of platelets to subendothelial surface: distinct from adhesion to collagen. Experientia 27,283. Born, G. V. R. (1973) Applicability of in vitro observations on the aggregation of platelets to their function in vitro. In Erythrocytes, Thrombocytes and Leukocytes (Edited by Gerlach, E., Moser, K., Deutsch, E. and Wilmanns, W.). Georg Thieme, Stuttgart. Brown, C. H. Leverett, L. B., Lewis, C. W., Alfrey, C. P., Jr. and HeUums, J. D. (1975) Morphological, biochemical and functional changes in human platelets subjected to shear stress. Journal of Laboratory and Clinical Medicine (in press).
Intravascular mural thrombi produced by acoustic microstreaming Glover, C. J., McIntire, L. V., Natelson, E. A. and Brown, C. H., III. Paper presented at the Amer. Inst. Chem. Eng. Meeting, Pittsburgh, Penn., 2-5 June, 1974. Henry, R. L. (1962) Methods for inducing experimental thrombosis. Angiology 13, 554. O'Brien, J. R. (1969) Cell membrane damage, platelet stickiness and some effects of aspirin. British Journal of Haematology 17, 610. Palko, P. D., Nanson, E. M. and Fedoruk, M. A. (1964) The early detection of deep vein thrombosis using ~3q-tagged human fibrinogen. Canadian Journal o[ Surgery 7, 215. Poliwoda, H., Deinhardt, J., Pluta, M., Hagemann, G. and Welling, H. (1973) Cinematographic investigations of the early phase of thrombus formation. In Erythrocytes, Thrombocytes and Leukocytes (Edited by Gerlach, E., Moser, K., Deutsch, E. and Wilmanns, W.). Georg Thieme, Stuttgart. Schiffmann, S. et al. (1973) Platelets and the initiation of intrinsic clotting. British Journal of Haematology 24, 633. Spilker, B. A. et al. (1973) Formation and embolisation of thrombi after electrical stimulation. On the method and evaluation of drugs. Thrombosis et diathesis haemorrhagica 30, 352. Thomas, D. P. (1972) The platelet contribution to arterial
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and venous thrombosis. Clinics in Haematology 1,267. Vermylen, J., DeGaetano, G. and Verstraete, M. (1973) Platelets and thrombosis. In Recent Advances in Thrombosis, p. 113. (Edited by Poller, L.). Churchill Livingstone, London. Wiedeman, M. P. (1973) Platelet aggregates induced by red blood cell injury. In Oxygen Transport to Tissue (Edited by Bruley, D. F. and Bicher, H. I.). Plenum Press, New York. Williams, A. R. (1972) Disorganisation and disruption of mammalian and amoeboid cells by acoustic microstreaming. J. Acoust. Soc. Am. 52, 688. Williams, A. R. (1974) Release of serotonin from human platelets by acoustic microstreaming. J. Acoust. Soc. Am. 56, 1640. Williams, A. R., Hughes, D. E. and Nyborg, W. L. (1970) Haemolysis near a transversely oscillating wire. Science 169, 871. Williams, A. R., O'Brien, W. D., Jr. and Coller, B. S. (1976) Exposure to ultrasound decreases the recalcification time of platelet rich plasma. Ultrasound Med. Biol. 2, 113. Williams, A. R., Sykes, S. M. and O'Brien, W. D., Jr. (1977). Ultrasonic exposure modifies platelet morphology and function in vitro. Ultrasound Med. Biol. 2, 311.
INTRAVASCULAR MURAL THROMBI PRODUCED BY ACOUSTIC MICROSTREAMING* A. R. WILLIAMS Department of Medical Biophysics, University of Manchester, Manchester, England. (First received 6 August 1976; and in final form 31 March 1977) Abstraet--A discrete portion of the wall of an intact blood vessel may be driven to oscillate at an ultrasonic frequency in vivo by the external application of a vibrating metal probe. The hydrodynamic forces generated within the local intravascular microstreaming field at the site of contact with the probe tip increase with increasing amplitude of oscillation of the ultrasonic driver. Under the appropriate experimental conditions one may produce (1) no detectable effects; (2) adhesion of platelets to apparently intact endothelium; (3) fibrin-free mural aggregates of platelets with the concomitant production of platelet micro-emboli; (4) mural thrombi of platelets permeated with fibrin and enveloped within a mixed clot and (5) obvious damage to the vessel wall and its endothelium with widespread clot formation. This technique might be of use in the in vivo screening of drugs which may interact with the haemostatic system. Key words: Acoustics, Ultrasonics, Ultrasonic damage, Platelet damage, Endothelial damage, Blood clot formation, Intravascular microstreaming. INTRODUCTION
Significant technical problems are associated with the production of discrete wellcharacterized thrombi at specific sites within the intact vascular tree of an experimental animal in vivo. If these thrombi are to be of use in the evaluation of the in vivo effectiveness of new pharmacologically active agents, which might promote or interfere with the haemostatic system, they have to be produced in a highly reproducible manner under carefully controlled experimental conditions. Numerous experimental systems have been proposed to produce adequate mural thrombi in vivo (Henry, 1962). These techniques are in general divisible into two main groups; one group damages and/or removes the endothelium (e.g. by pinching a vessel with fine forceps; electrical coagulation of a portion of a vessel wall (Spilker et al., 1973) or by the insertion of a rigid probe or inflatable balloon to scour the vessel wall (Palko et al., 1964; Baumgartner et al., 1971)) while the other group modifies the chemical environment adjacent to the endothelium so that platelets are attracted to it and adhere (e.g. by the electrodynamic diffusion of platelet aggregating agents through the vessel wall, (Born, I973) or the thermal precipitation of erythrocyte contents by means of a finely *Work accomplished under the partial support of contract number FDA 3177-75 (J) from the Bureau of Radiological Health, United States Food and Drug Administration.
focussed laser beam (Poliwoda et al., 1973; Wiedeman, 1973)). Platelets contribute to clot formation in the above experimental models by first adhering to "altered" endothelium or exposed subendothelial structures and subsequently by participating in and accelerating the conversion of fibrinogen to fibrin (Thomas, 1972; Vermylen et al., 1973). However, platelets may not have to adhere to a "damaged" endothelial surface before they are stimulated to participate in clot formation (Schiffman et al., 1973). Human platelets are extremely susceptible to damage by hydrodynamic shear stresses and undergo membrane rupture and the loss of intracellular components when exposed to stresses greater than 50 to 100 dyn cm -2 for 5 min (Brown et al., 1975). Similarly, Williams (1974) has demonstrated rupture of the platelet plasma membrane and the release of incorporated serotonin after millisecond exposures to shear stress within the acoustic microstreaming field generated around a steel wire oscillating transversely at 20kHz. Rupture of the plasma membrane increased platelet "stickiness" (i.e. that tendency of platelets to adhere to adjacent surfaces and to participate in spontaneous aggregation) (O'Brien, 1969) and increased the availability of platelet procoagulant agents (PF-3) which subsequently accelerated the rate of fibrin formation in vitro (Glover et al., 1975; Williams et al., 1976, 1977). Thus platelets which have suffered mem191
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brane damage following exposure to hydrodynamic shear stress are more adhesive than their controls and may be already "primed" to participate in fibrin formation. This paper describes a novel technique whereby platelets within the fluid blood are damaged by hydrodynamic streaming forces while adjacent to the vascular endothelium so that they might themselves initiate formation of a discrete mural thrombus in vivo without recourse to prior "alteration" or damage to the vascular endothelium.
MATERIALS AND METHODS
The ultrasonic driver consists of a short resonant length (1.69cm) of ll5/~m radius steel wire clamped at one end to the tip of a stainless steel velocity transformer. This velocity transformer was bonded with epoxy resin to a PZT 4 ultrasonic transducer and driven to oscillate in its longitudinal mode at 20 kHz. The wire was clamped at right angles to the long axis of the transducer/velocity transformer so that a flexural (transverse) standing wave was set up along the length of the wire (Williams et al., 1970). Thus, portions of the wire were driven to oscillate transversely at 20kHz in a plane between the limits indicated diagrammatically by the broken lines in Fig. 1. The amplitude of transverse oscillation of the wire increased as the power to the ultrasonic transducer was increased. At all times, the maximum value of transverse oscillation was found at the hemispherical tip of the free end of the wire; all displacement amplitude values quoted in this article (up to the maximum of about 30/xm) refer to the displacement amplitude measured at this free end of the wire in its unloaded condition (see Discussion). The transducer assembly was firmly mounted in rubber and held so that the tip of the wire was positioned at the focus of a low power (up to x30) steroscopic dissecting microscope. When the hemispherical tip of the oscillating wire is in contact with a fluid medium it acts as a local generator of small scale (d.c.) acoustic microstreaming (Williams et al., 1970; Williams, 1974). This localized streaming field is seen as symmetrical eddy currents of fluid immediately adjacent to the wire tip. Surface waves (ripples) were visible when the liquid meniscus met the wire at any point other than at a displacement node. These ripples were also seen in the thin film
P iii
I
iI x
w) i
Fig. 1. A diagrammatic representation of the sonication system. A resonant length of steel wire (W) was clamped at right angles to the tip of a steel probe (P) oscillating in its longitudinal mode at 20kHz. This configuration produced a flexural (transverse) standing wave pattern along the length of the wire as indicated by the broken lines, which represent the greatly exaggerated limits of displacement amplitude. The hemispherical tip of the free end of the wire was pressed against the outside wall of an exposed but intact blood vessel in vivo and driven so that a portion of the vessel wall was made to oscillate with the wire tip.
of saline bathing the exposed blood vessels and were used to "tune" the transducer assembly so that the wire was always oscillating with maximum displacement amplitude for that particular value of applied electrical power. Young adult male mice (Manchester strain of albino Swiss) were anaesthetized with Nembutal (Pentobarbital) at a dosage of 60mg/kg administered intraperitoneally. A flap of skin over the inside of the thigh was carefully resected to expose the femoral artery and vein, and the transparent thin fibrous sheet (the fascia lata) which overlies the vessels was not disturbed. The mouse was placed on a thermostated microscope stage which was raised until the fascia lata over the chosen blood vessel pressed against the wire tip (Fig. 1). The terminal portion of the leg under investigation was attached to a micromanipulator to damp out spontaneous movements of the limb and enable minute adjustments to be made in the height and position of the blood vessel relative to the wire tip (which remained at the focus of the dissecting microscope). The height of the limb was adjusted until the wire tip made a visible indentation in the vessel wall (i.e. it protruded between about 20 and 50% of the vessel diameter). This indentation was approximately conical in shape and resembled the indentation made by
Intravascular mural thrombi produced by acoustic microstreaming pushing a finger into an air-filled balloon. The hemispherical wire tip was the only portion of the wire physically in contact with the vessel wall. This wall was held in intimate contact with the wire tip by a small elastic restoring force from the deformed membrane and by the intraluminal pressure generated within the intact vessel by the heart of the animal (i.e. by its own blood pressure). This pressure ensured good coupling so that the portion of the vessel wall in contact with the wire tip was also driven to oscillate at 20 kHz. The oscillating portion of the vessel wall then acted as a local generator of acoustic microstreaming within the fluid blood (Fig. 2). The exposed blood vessels were kept moist by the repeated application of small quantities of isotonic saline at a temperature of 38°C. At the end of the sonication procedure, a small drop of an inert colouring material (India ink) was allowed to run down the wire to mark the site of irradiation. Once dry, the mark remained as a discrete spot despite repeated washings with various fixatives and staining reagents, and did not appear to alter the morphology of the underlying tissues. After irradiation, the blood of the animal was allowed to flow through the exposed vessels for various times within the range 0 to 3000 sec before they were tied off with surgical sutures and fixed or frozen in situ. Vessels which had been tied off with surgical sutures and partially fixed in situ with isotonic aldehydes were excised, fixed, dehydrated and embedded in paraffin wax. Alternatively, vessels were tied off and
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frozen in situ with isopentane which had been cooled in liquid nitrogen, excized, mounted in liver on a metal block and sectioned in a Slee cryostat. Thin sections were stained with Haematoxylin/Eosin, Masson's Trichrome or M.S.B. (which stains fibrin red and platelets blue). The freezing and thawing associated with obtaining frozen sections, combined with the low osmotic strength of the first stage of the staining procedure for Masson's trichrome (aqueous Haematoxylin), lysed the erythrocyte population. Samples destined for electron microscopy were tied off with surgical sutures and bathed in isotonic cacodylate buffer pH 7.4 containing 2% Glutaraldehyde for 15 min. The rigid vessels were excized, fully fixed in the same fixative for 1 hr, post fixed in osmium tetroxide, dehydrated and embedded in e p o x y resin (AralditeR). Thin sections were stained with uranyl acetate and lead nitrate and examined in a Phillips EM201 electron microscope. RESULTS
A separate series of experiments were undertaken to investigate the effect of contact pressure between the wire tip and the vessel wall on the results presented below. An adult male mouse was anaethetized and a portion of its small intestine with its intact mesenteric circulation was placed on a heated (38°C) microscope stage. A 300 to 350/xm diameter blood vessel was transilluminated and the transversely oscillating wire (mounted on a micromanipulator) was advanced until it made contact with the vessel wall. The wire was driven at a maximum tip displace-
Fig. 2. This diagram shows the relative dimensions of the structures at the site of contact with the wire (W)tip. The vessel wall (V) was displaced by a static contact force a distance, d, which was maintained between 20 and 50% of the vessel diameter (D). When the vessel wall was driven to oscillate, symmetrical eddies of acoustic microstreaming could be seen within the intact blood vessel, as indicated by the broken lines.
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ment amplitude of 8 or 12/~m. The depth of penetration of the wire tip into the intact blood vessel (expressed as a percentage of the vessel diameter) was taken as an indirect estimate of the contact pressure since a more direct measurement was not available. Under the appropriate illumination conditions, intravascular acoustic microstreaming is seen as two symmetrical dark regions approximately egg-shaped situated on either side of the mid point of contact with the wire tip. The diameter of these "shadows" increases rapidly with increasing penetration of the wire over the range 0 to about 10 or 15% of the vessel diameter; the wire displacement amplitude being held constant. Further penetration of the wire up to about 60% of the vessel diameter had no visible effect on the microstreaming patterns (Fig. 2). Deeper penetrations were not attempted. The histological results reported below were obtained from mouse femoral arteries and veins where the intravascular microstreaming eddies could not be seen. Consequently, histological sections were cut from femoral arteries and veins from different legs of the same mouse after they had been irradiated at the same wire displacement amplitude (8 or 12/~m) for the same time (5 min) but at 20 or 50% penetration of the vessel diameter. The results obtained were sufficiently similar to indicate that the extent of wire penetration within this range had no detectable effect on the histological appearance of the vessel. There was also no sign of thermal damage at the site of contact with the wire tip. Light and electron microscopical examination of the vessel wall at the site of contact with the wire tip showed transverse wire displacement amplitudes of the order of 2 to 4 p~m at 20 kHz produced no damage to the vascular endothelium which could not have been caused by the mechanical trauma associated with freezing, cutting, staining or washing the thin sections. However, some histological changes were observed at wire displacement amplitudes greater than 5/~m. In general, the nature and extent of the observed changes was influenced by the displacement amplitude of the wire, the duration of the ultrasonic exposure and the time interval between exposure and fixation in situ prior to removal for histological examination. The role of the vascular endothelium in thrombus formation following ultrasonic
exposure was investigated by means of the electron microscope. Preliminary results showed that occasional platelets (PI) had fused with the endothelium of mouse femoral veins at the site of contact with the wire tip after it had been driven at displacement amplitudes of the order of 5 to 8 ~m for 5 min (Fig. 3). These fused platelets had undergone the release reaction (i.e. were degranulated), contained bundles of microtubules (arrowed) which extended into projections from the platelet surface and frequently exhibited large-scale rupture of the plasma membrane with loss of cytoplasmic material. Each fused platelet had one or more intact or damaged platelets (P2) adhering to it. Apart from these occasional fused platelets (approximately one for each 50 endothelial cells viewed as thin sections cut in a plane at right angles to the long axis of the vessel) there was no obvious damage to the endothelium, which remained apparently intact and full of pinocytotic vesicles (Fig. 3). Light micrographic investigations of frozen and wax embedded blood vessels which had been sonicated at wire displacement amplitudes in the range 10 to 15 ~m showed the presence of mural thrombi at the site of contact with the wire tip. The size and type of mural thrombus varied with the amplitude of oscillation of the ultrasonic driver and the time interval between irradiation and fixation. Mouse femoral veins which had been subjected to a wire displacement amplitude of about 10/.~m for 5 min and then left for a further 20 min with blood flowing over the irradiated area produced large "loose" mural aggregates of platelets (Fig. 4). These large aggregates could achieve a diameter of up to 05 mm and a length of several millimetres. Histologically, these mural thrombi consisted of a core of densely packed (but not fused) platelets surrounded by a zone of loosely adhered platelets, the whole being infiltrated with leucocytes. There was no visible evidence of fibrin formation or erythrocyte participation (Fig. 4). Figure 5 is light photomicrograph of a longitudinal cryostat (frozen) section of a mouse femoral vein stained by the trichrome procedure (which has lysed the erythrocyte population). The oscillating wire was positioned upstream of the field of view and driven at a displacement amplitude of 12 ~m for 20 min. A large "loose" mural aggregate of platelets similar to that depicted in Fig. 4 was sub-
Intravascular mural thrombi produced by acoustic microstreaming
Fig. 3. This electron micrograph shows the intact and apparently undamaged endothelium (E) of a mouse femoral vein which had been subjected to a wire displacement amplitude of about 8/xm for 5 min. The platelet Pm (which has suffered extensive rupture and removal of its plasma membrane and the loss of some intracellular organelles) has attached itself to or fused with the endothelium where it has attracted another platelet (P2) which has adhered to it forming a micro-mural aggregate. The arrow indicates the position of a bundle of microtubules. (1/~m = 2.64 cm.)
Fig. 4. Light micrograph of a transverse cryostat (frozen) section through a mouse femoral vein (V) which had been irradiated at 10 tzm for 5 min and blood left to flow through the vessel for a further 20 min before fixation and removal. The lumen of the vessel was largely occluded by a "loose" mural aggregate of fibrin-free platelets (P) infiltrated with leucocytes. Erythrocytes were lysed during the staining procedure with Masson's trichrome. (100 ~m = 1.25 cm.)
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Fig. 5. Longitudinal cryostat section through a mouse femoral vein stained with Masson's trichrome. The wire was placed upstream of the field of view and driven at an amplitude of about 12 p.m for 20 min where it produced a "loose" mural aggregate of platelets similar to that seen in Fig. 3. The vessel was frozen with cold isopentane while the wire was still oscillating. Numerous fibrin-free micro-emboli of platelets (A) can be seen en r o u t e to the heart and lungs through the valve (V). The arrow indicates the direction of blood flow. (100 ~m = 2.18 cm.)
Fig. 6. A transverse section through the femoral artery (A) and vein (V) of a mouse after the wire had been applied to the fascia lata overlying the inflated vein (marked with India ink, I) and driven at an amplitude of about 20/zm for 5 rain. A compact mural aggregate of platelets (T) is seen within a mixed clot on that portion of the wall which, when inflated, was closest to the wire tip. The vessels were fixed in situ, embedded in paraffin wax and thin sections stained with Haematoxylin/Eosin. (100 krm = 1.2 cm.)
Intravascular mural thrombi produced by acoustic microstreaming
Fig. 7. Another section at a higher magnification of the same mural thrombus presented in Fig. 6, but stained by the M.S.B. procedure to demonstrate fibrin. A dense band of fibrin (F) is visible at the endothelial junction from which fibrin strands permeate the platelet aggregate (A). The arrow indicates a mixed thrombus consisting predominantly of erythrocytes enmeshed in a fibrin network. (40/~m = 1.88 cm.)
Fig. 8. A longitudinal cryostat section through a mouse femoral vein (V) which had been completely thrombosed following 5 min irradiation at a wire displacement amplitude of about 30 ~m. The wire was placed upstream of the field of view where it had damaged the vascular endothelium. The lumen of the vessel contains a densely staining fibrous mass (F) consisting mainly of fibrin embedded in a cytoplasmic material which may once have been platelets, and a homogeneous background material (B) which resembles a typical fresh mixed clot after the erythrocytes have been lysed by the staining procedure. (100 tim = 1.84 cm.)
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Intravascular mural thrombi produced by acoustic microstreaming
sequently found at the site of contact with the wire tip. However, in this case the vessel was not tied off and fixed in situ but merely clamped shut with forceps while the wire was still oscillating. Isopentane, cooled in liquid nitrogen, was poured on to the exposed vessel as soon as it was clamped; this rapidly froze the blood and maintained the correct spatial relationship of the intravascular contents. Numerous small "loose" platelet aggregates (A) can be seen floating freely within the lumen of the vessel, frequently in association with leucocytes (Fig. 5). These micro-aggregates are visible on both sides of the venous valves (V) and are presumably being borne by the circulation to the heart and lungs. Mural thrombi produced near the wire tip at higher displacement amplitudes (e.g. 15 to 20 tzm) were more compact than those described above, and were frequently found in association with erythrocytes. Figure 6 is a low power light micrograph of a section through the femoral artery (A) and femoral vein (V) of a young adult male mouse, stained with Haematoxylin/Eosin. India ink (I) marks the site at which the wire tip was applied to the fibrous fascia lata which was in contact with the femoral vein when it was fully inflated with blood. The thin muscular coat of the vein was not enough to maintain its circular cross-section when the vessel was cut and embedded in paraffin wax. The wire was driven at a displacement amplitude of about 20 txm for 5 min, and blood was allowed to flow over the irradiated region for 20min before the vessel was tied off and fixed in situ with formal saline. Most of the free blood cells (i.e. those cells not attached to the vessel walls or participating in thrombus formation) were lost during the fixation, embedding, sectioning and staining procedures. A mural thrombus (T) consisting of tightlypacked platelets was visible on that portion of the wall immediately adjacent to the site of contact with the wire tip (Fig. 6). Serial sections showed that this thrombus was at least 3 mm long. The platelet thrombus was enclosed within a mixed thrombus consisting mainly of erythrocytes. Another mixed thrombus was found on the opposite wall of the same vessel, i.e. the wall which was furthest away from the site of contact with the wire tip, but this thrombus did not contain a region consisting predominantly of platelets.
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Figure 7 is a light micrograph of another section of the same mural thrombus of tightly packed platelets presented in Fig. 6, but taken at a higher magnification. This section was stained by the M.S.B. procedure which specifically stains fibrin bright red while platelets appear blue. A dense red band was visible at the platelet/endothelium junction which merged into the remainder of the platelet mass which was lilac/purple in colour and permeated with thin lines of red. Strands of fibrin could also be seen amongst the erythrocytes at the periphery of the platelet thrombus. Blood vessels irradiated at wire displacement amplitudes greater than about 20 ~m showed immediate signs of damage. Blood was observed to seep from the vessels into the isotonic saline bathing fluid. Gross histological damage was observed in both the muscular walls of the irradiated vessels and the vascular endothelium. The flow of blood within the vessels was grossly perturbed by the formation of two large rapidly-rotating vortices which completely filled the lumen of the vessel. Femoral veins having a diameter of up to 2 mm could be completely occluded by thrombus formation within a few minutes of irradiation at a wire displacement amplitude of the order of 30 tzm. Figure 8 is a representive light micrograph of a cryostat (frozen) section stained with Masson's trichrome of a mouse femoral vein (V) which had been completely occluded by a thrombus. The wire was placed upstream of the field of view and driven at a displacement amplitude of about 30/zm for 5 min. The direction of blood flow was determined from the orientation of the vessel on the metal mounting block in the cryostat and the appearance of valves within the veins. The vessel contents are clearly divisible into two components: one is the homogeneous background material (B) which consists of erythrocyte ghosts (lysed by the staining procedure) and platelets bound together with fibrin and the other is a dense fibrous material (F) which appears to consist mainly of fibrin and a cellular material which may once have been platelets (Fig. 8). DISCUSSION
Symmetrical intravascular eddy currents may be seen within the blood adjacent to the tip of a transversely oscillating wire after it has been applied to the outside of an intact
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mesenteric blood vessel in vivo. This intravascular streaming appears to be identical with the acoustic microstreaming field which is generated when the same oscillating wire is immersed in a homogeneous fluid (Williams et al., 1970). This indicates that a discrete portion of the blood vessel has been driven to oscillate at an ultrasonic frequency (20 kHz) so that it acts as a local generator of smallscale acoustic microstreaming within the fluid blood. The only practicable measurement of the contact force between the wire and the inflated blood vessel was the depth of penetration of the wire tip (expressed as a percentage of the vessel diameter). It has been determined experimentally that both the visual appearance of the microstreaming eddies and their histological sequelae were virtually identical whether the penetration depth was 20 or 50% of the vessel diameter. These observations, together with the absence of detectable thermal damage to the superficial vascular structures in contact with the wire tip, indicate that the wire was efficiently coupled to the vessel wall. The displacement amplitude of the wire tip could not be measured during the course of the experiment as it was buried within the lumen of the vessel. The frequent occurrence of slow spontaneous limb movements meant that the operator was fully employed maintaining the correct depth of wire penetration and ensuring that the wire was oscillating with maximum efficiency for that value of applied electrical power (as indicated by surface waves on the saline bathing the contact site). The displacement amplitudes quoted below were measured at the tip of the wire when the same driving voltage was applied to the piezoelectric transducer, but with the wire in an unloaded condition (i.e. the wire tip was not in contact with the vessel wall). There was no significant change in the displacement amplitude measured at the antinode adjacent to the wire tip when that tip was loaded by contact with an inflated blood vessel. Similar indirect measurements suggest that the increased damping resulting from contact with the vessel wall decreased the displacement amplitude of the tip by less than 10%. Thus, the displacement amplitude values quoted in the text represent the maximum limits of oscillatory motion which could only be obtained within the vessel wall under
conditions of zero damping and 100% efficient coupling. The biological results obtained at each discrete value of transverse wire displacement amplitude are influenced by the duration of the ultrasonic exposure and the time interval between exposure and removal of the vessel for histological examination. Nevertheless, it is convenient to partition the results obtained into five overlapping compartments or groups on the basis of increasing wire displacement amplitude (since this is a measure of this instantaneous hydrodynamic shear stress generated at the blood/endothelial cell interface). Briefly, the groups are: (I) no detectable effects; (2) occasional platelet adhesion to apparently normal endothelium; (3) "loose" mural aggregates of fibrin-free platelets with the concomitant production of platelet microemboli; (4) dense mural thrombi of platelets containing fibrin and associated with mixed blood clots and (5) gross damage to the vessel wall and widespread clot formation which occludes even major blood vessels. Group 1 includes those results obtained at wire displacement amplitudes below about 4t~m at 20kHz where no perturbation of blood flow was observed and histological sections were indistinguishable from their controls. Surface waves were clearly visible on the thin film of saline bathing the outside surface of the vessel during irradiation showing that wire was oscillating. This implies either that the intravascular streaming forces produced at these low wire amplitudes were not large enough to perturb the normal blood flow and damage cellular elements, or that the oscillatory energy was dissipated as "slip" or by the deformation of elastic structures within the vessel wall. Group 2 includes those results obtained between wire displacement amplitudes of 5 and about 8 ~m after 5 min irradiation at 20 kHz. These samples appeared normal with the light microscope but preliminary electron micrographic examination of the site of contact with the wire tip showed that occasional platelets had adhered or fused with what appeared to be normal endothelial surface (Fig. 3). Many of the fused platelets (P1) showed evidence of large-scale membrane rupture and the loss of cytoplasmic structures including secretory granules, mitochondria and dense bodies. Each fused platelet had one or more other platelets (P2) adhering to it forming a micro-mural thrombus (Fig. 3). It
Intravascular mural thrombi produced by acoustic microstreaming
was not possible to determine from the electron micrographs whether the platelet plasma membrane was ruptured before or after it became adhered to the endothelial surface. Two conflicting hypotheses may be proposed to explain the mechanism of this crucial interaction between platelets and endothelium. One is that the intravascular acoustic microstreaming field produced many minute lesions at discrete sites on the endothelial surface. Normal platelets would then be attracted to these sites where they would undergo the release reaction and adhere. If it is assumed that the plasma membrane of each endothelial cell is of uniform susceptibility to ultrasound, then the hydrodynamic streaming forces generated by the microstreaming field ought to produce a uniform region of endothelial cell damage over the entire area adjacent to the wire tip (4.3 × 104/zm2) and not merely at discrete sites. However nonuniformity of the membrane could lead to localized damage at susceptible sites. An alternative hypothesis assumes that the platelet plasma membrane was ruptured before it became adhered to the endothelium. There is no precedent for presuming the spontaneous rupture of the platelet plasma membrane immediately following its adhesion to the endothelium, and yet damaged platelets have been found attached to the endothelial cells immediately after irradiation, suggesting that they were damaged before they became attached. Human platelets are notoriously susceptible to damage by hydrodynamic shear stress (see Introduction) and undergo large scale rupture of the plasma membrane when subjected to the acoustic microstreaming field generated around a steel wire oscillating transversely at 20 kHz (Williams, 1974). The electron microscopic appearance of human and canine platelets which have been partially disrupted in this way closely resemble the mouse platelets PI and P2 depicted in Fig. 3, i.e. most of the intracellular contents remain bound together even though a portion of the plasma membrane has been removed (Williams et al., 1977). The intracellular contents of mouse ascites cells also remain firmly adhered together when portions of the plasma membrane are torn away by the same acoustic microstreaming field (Williams, 1972). It is, therefore, not unreasonable to propose that the same adhesive forces which bind the intracellular components together in mouse platelets might
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(when exposed following membrane rupture) also adhere that same platelet remnant to (undamaged) endothelium. The obvious "stickiness" of ultrasonically generated platelet emboli supports this proposal, as does the absence of re-growth of a mural aggregate following macro-embolization, when the wire has ceased to oscillate. It is of interest that this threshold for mouse platelet damage in vivo (5 to 8/~m) agrees well with the minimum value of about 6 ~m for the release of incorporated serotonin from human platelets irradiated in vitro with the same transversely oscillating wire assembly (Williams, 1974). This confirms the extreme shear sensitivity of platelets since these values correspond to hydrodynamic shear stresses of the order of 100 to 250 dyn cm -2. This result also indicated that at these displacement amplitudes the damping effect of the vessel wall on the oscillatory motion of the wire is negligible and that coupling losses can be ignored. Group 3 results were obtained at wire displacement amplitudes between about 10 and 15/zm. Large mural thrombi of platelets infiltrated with leucocytes were usually found at the site of contact with the wire tip (Fig. 4). These mural aggregates invariably had a "loose" appearance in histological sections with no visible evidence of fibrin formation or erythrocyte involvement. Each mural aggregate "shed", or initiated the local formation of micro-aggregates of fibrin-free platelets which were embolized downstream both during and after the period of irradiation. A sample of this "snowflake-like" stream of platelet micro-emboli is presented in Fig. 5. The growing platelet thrombus occasionally became dislodged from the vessel wall and embolized downstream. Frequently, there was no visible re-growth of a mural thrombus at that site in the absence of the ultrasonic field. Group 3 results appear to be an extension of Group 2 results (i.e. adhesion of platelets to the endothelium) with the additional selfperpetuating cycle of platelet attraction, release, adhesion to the growing aggregate and the attraction of more platelets. This rapid growth without detectable fibrin formation explains the large size and "loose" consistency of Group 3 thrombi and their tendencies to shed or initiate micro-emboli and themselves become dislodged as macroemboli. The frequent absence of re-growth (in the absence of the acoustic microstream-
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A. R. WILLIAMS
ing field) indicates that the endothelium at the site of contact with the wire tip has suffered minimal damage. Group 4 results were obtained at wire displacement amplitudes between about 15 and 20/zm. The mural aggregates of platelets were invariably smaller and more compact than those of Group 3 and were usually surrounded by a mixed thrombus consisting predominantly of erythrocytes (Figs 6 and 7). Mixed thrombi without a central "core" of platelets were frequently found attached to other portions of the vessel wall. Figure 7 is another section of the same mural thrombus presented in Fig. 6 but stained by the M.S.B. procedure to demonstrate the presence of fibrin. The tightly-packed platelet aggregate was seen to be permeated with fibrin strands which appeared to have grown out of the dense band of fibrin which was present at the thrombus-endothelial junction. These fibrin strands were also visible between the erythrocytes enveloping the mural thrombus of platelets (Fig. 7). Group 4 results appear to be similar to those of Group 3 with the exception that there has been the rapid and extensive production of fibrin. The platelet/fibrin mural thrombus has contracted and mixed clot formation has been initiated at its surface. Any portion of the vessel where temporary blood stasis could occur was usually filled with a mixed thrombus which attached itself to the vessel wall (the region diametrically opposite the point of application of the wire tip was one such region). Group 5 results were obtained at wire displacement amplitudes greater than about 20 mm and represent a more extreme version of the results presented in Group 4. The vessel wall and its endothelium were obviously damaged by this large oscillatory motion of the wire and so the effects of the normal haemostatic mechanism of the animal were superimposed upon the effects produced by the acoustic microstreaming field. Complementary information was obtained from visual observation of thrombus induction and sealing within mouse mesenteric vessels which were transilluminated on a heated microscope stage. Mural aggregates grew very rapidly (in less than a second) at these large wire displacement amplitudes and could frequently be seen issuing as a continuous fluid stream of platelets from the site of irradiation. This flexible gelatinous mass of
aggregated (and partially disrupted) platelets is extremely "sticky" and adhered itself to the endothelial surface at every contact. Simultaneously, fibrin formation is occurring within the platelet aggregate rendering it progressively more rigid and securing its attachment to the vessel wall. The fibrous material (F) in Fig. 8 appears to be the solidified remains of the original fluid platelet thrombus (now completely permeated with fibrin) which has become attached at all points around the vessel circumference. The blood stasis induced by this solidifying "plug" enabled fibrin formation to occur within the fluid blood giving the mixed clot seen as the homogeneous background material (B) in Fig. 8. Thus, different facets of the haemostatic mechanism as it operates in vivo may be selectively produced at adjacent sites within the same experimental animal merely by the choice of the appropriate wire displacement amplitude. For any given animal anaesthetized by the method of choice, it should be possible to determine the optimum combination of amplitude, frequency of oscillation and duration of exposure which is required to produce no detectable effect (Group 1), platelet adhesion without apparent aggregation (Group 2), mural aggregates without detectable fibrin formation (Group 3), mural aggregates with fibrin formation (Group 4) and, finally, gross damage to the vessel wall and its endothelium with widespread thrombus formation (Group 5). This technique ought, therefore, to be of use in the in vivo screening of pharmacologically active agents which might enhance or interfere with various aspects of the complex haemostatic system. Acknowledgements--I wish to thank Mr John Sherwin for his invaluable technical assistance, Mrs W. Staley and Mr R. Ellis for the photography, Miss N. Osborn for the artwork and Miss K. Holt for typing the manuscript. REFERENCES Baumgartner, H. R., Stemerman, M. B. and Spaet, T. H. (1971) Adhesion of platelets to subendothelial surface: distinct from adhesion to collagen. Experientia 27,283. Born, G. V. R. (1973) Applicability of in vitro observations on the aggregation of platelets to their function in vitro. In Erythrocytes, Thrombocytes and Leukocytes (Edited by Gerlach, E., Moser, K., Deutsch, E. and Wilmanns, W.). Georg Thieme, Stuttgart. Brown, C. H. Leverett, L. B., Lewis, C. W., Alfrey, C. P., Jr. and HeUums, J. D. (1975) Morphological, biochemical and functional changes in human platelets subjected to shear stress. Journal of Laboratory and Clinical Medicine (in press).
Intravascular mural thrombi produced by acoustic microstreaming Glover, C. J., McIntire, L. V., Natelson, E. A. and Brown, C. H., III. Paper presented at the Amer. Inst. Chem. Eng. Meeting, Pittsburgh, Penn., 2-5 June, 1974. Henry, R. L. (1962) Methods for inducing experimental thrombosis. Angiology 13, 554. O'Brien, J. R. (1969) Cell membrane damage, platelet stickiness and some effects of aspirin. British Journal of Haematology 17, 610. Palko, P. D., Nanson, E. M. and Fedoruk, M. A. (1964) The early detection of deep vein thrombosis using ~3q-tagged human fibrinogen. Canadian Journal o[ Surgery 7, 215. Poliwoda, H., Deinhardt, J., Pluta, M., Hagemann, G. and Welling, H. (1973) Cinematographic investigations of the early phase of thrombus formation. In Erythrocytes, Thrombocytes and Leukocytes (Edited by Gerlach, E., Moser, K., Deutsch, E. and Wilmanns, W.). Georg Thieme, Stuttgart. Schiffmann, S. et al. (1973) Platelets and the initiation of intrinsic clotting. British Journal of Haematology 24, 633. Spilker, B. A. et al. (1973) Formation and embolisation of thrombi after electrical stimulation. On the method and evaluation of drugs. Thrombosis et diathesis haemorrhagica 30, 352. Thomas, D. P. (1972) The platelet contribution to arterial
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and venous thrombosis. Clinics in Haematology 1,267. Vermylen, J., DeGaetano, G. and Verstraete, M. (1973) Platelets and thrombosis. In Recent Advances in Thrombosis, p. 113. (Edited by Poller, L.). Churchill Livingstone, London. Wiedeman, M. P. (1973) Platelet aggregates induced by red blood cell injury. In Oxygen Transport to Tissue (Edited by Bruley, D. F. and Bicher, H. I.). Plenum Press, New York. Williams, A. R. (1972) Disorganisation and disruption of mammalian and amoeboid cells by acoustic microstreaming. J. Acoust. Soc. Am. 52, 688. Williams, A. R. (1974) Release of serotonin from human platelets by acoustic microstreaming. J. Acoust. Soc. Am. 56, 1640. Williams, A. R., Hughes, D. E. and Nyborg, W. L. (1970) Haemolysis near a transversely oscillating wire. Science 169, 871. Williams, A. R., O'Brien, W. D., Jr. and Coller, B. S. (1976) Exposure to ultrasound decreases the recalcification time of platelet rich plasma. Ultrasound Med. Biol. 2, 113. Williams, A. R., Sykes, S. M. and O'Brien, W. D., Jr. (1977). Ultrasonic exposure modifies platelet morphology and function in vitro. Ultrasound Med. Biol. 2, 311.