(Circulation. 2000;102:531.)
© 2000 American Heart Association, Inc.
Clinical Investigation and Reports |
Noninvasive Imaging of Inflammation by Ultrasound Detection of Phagocytosed Microbubbles
From the Cardiovascular Division (J.R.L., M.P.C., S.K.) and the Department of Biomedical Engineering (P.A.D., K.L., J.S., S.K., K.F.), University of Virginia School of Medicine, Charlottesville.
Correspondence to Jonathan R. Lindner, MD, Box 158, Cardiovascular Division, University of Virginia Medical Center, Charlottesville, VA 22908. E-mail jlindner{at}virginia.edu
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Abstract |
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Background—We have previously shown that microbubbles adhere to leukocytes in regions of inflammation. We hypothesized that these microbubbles are phagocytosed by neutrophils and monocytes and remain acoustically active, permitting their detection in inflamed tissue.
Methods and Results—In vitro studies were performed in which
activated leukocytes were incubated with albumin or
lipid microbubbles and observed under microscopy. Microbubbles attached
to the surface of activated neutrophils and monocytes, were
phagocytosed, and remained intact for up to 30 minutes. The rate of
destruction of the phagocytosed microbubbles on exposure to ultrasound
was less (P
0.05) than that of free microbubbles at all
acoustic pressures applied. Intravital microscopy and
simultaneous ultrasound imaging of the cremaster muscle was
performed in 6 mice to determine whether phagocytosed microbubbles
could be detected in vivo. Fifteen minutes after
intravenous injection of fluorescein-labeled
microbubbles, when the blood-pool concentration was negligible, the
number of phagocytosed/attached microbubbles within venules was 7-fold
greater in tumor necrosis factor-
(TNF-)–treated animals than in
control animals (P<0.01). This increase in retained
microbubbles resulted in a 5- to 6-fold-greater
(P<0.01) degree of ultrasound contrast enhancement than
in controls.
Conclusions—After attaching to activated neutrophils and monocytes, microbubbles are phagocytosed intact. Despite viscoelastic damping, phagocytosed microbubbles remain responsive to ultrasound and can be detected by ultrasound in vivo after clearance of freely circulating microbubbles from the blood pool. Thus, contrast ultrasound has potential for imaging sites of inflammation.
Key Words: imaging • inflammation • ultrasonics • leukocytes
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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We have recently demonstrated that microbubbles attach to activated leukocytes that are adherent to the endothelium of postcapillary venules after ischemia-reperfusion and during tumor necrosis factor-
(TNF-)–induced inflammation.1 The extent of
microbubble attachment correlates with the degree of
inflammation.1 This phenomenon is probably responsible for
the prolonged myocardial opacification seen after the administration of
microbubbles after myocardial ischemia and
reperfusion.2 We have shown that microbubble binding to leukocytes is influenced by the composition of the microbubble shell. Microbubbles composed of albumin adhere primarily through the leukocyte ß2-integrin Mac-1 (CD11b/CD18), whereas lipid microbubbles adhere through opsonization by serum complement.1 The cell types responsible for leukocyte-microbubble interaction and the fate of microbubbles after their attachment to activated leukocytes is unknown.
In this study, we hypothesized that microbubbles are phagocytosed by activated leukocytes that express Mac-1 and other complement receptors and remain acoustically active, thereby permitting their detection at sites of inflammation. To investigate these hypotheses, in vitro studies were performed to determine the leukocyte cell types involved and to assess the responses of phagocytosed microbubbles to ultrasound (US). The feasibility of imaging these microbubbles in vivo was assessed by means of simultaneous intravital microscopy and US imaging of inflamed cremaster muscle in mice.
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Methods |
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Microbubbles and Isolation of Leukocytes
Perfluorocarbon gas–filled microbubbles with shells composed of either albumin or lipid (Optison or MP1950, Mallinckrodt Medical) were used. Lipid microbubbles contained phosphatidylcholine, a surfactant, and a fluorescein derivative of phosphatidylethanolamine in a molar ratio of
75:15:1. The
microbubble size ranged from 2.8 to 4.1 µm. Approximately
2x107 microbubbles were used for each in vitro
experiment. Leukocytes were isolated from whole blood collected from healthy volunteers. A portion of this blood was used to obtain serum and the remainder was anticoagulated with heparin (10 U/mL). Neutrophil and monocyte/lymphocyte fractions were isolated with Ficoll-Hypaque density gradient centrifugation (Mono-Poly, ICN Pharmaceuticals),3 washed twice, and resuspended in PBS. Leukocyte concentrations were determined with hemocytometer measurements of Kimura-stained samples. For each in vivo experiment, 2x106 leukocytes were combined with 0.2 mL of serum and 0.2 mL of PBS containing 2 mmol/L MgCl2 and CaCl2. Leukocytes were activated by 10 nmol/L phorbol myristate acetate (PMA) 15 minutes before use.
Determination of Cell Types Responsible for
Leukocyte-Microbubble Interactions
Flow cytometry was performed to determine the cell types
involved in leukocyte-microbubble interactions. Activated or
nonactivated leukocytes were combined with
fluorescein-labeled albumin or lipid microbubbles
and incubated during gentle agitation for 3 minutes at 37°C. Red
blood cells were hypotonically lysed, and samples were analyzed
with a flow cytometer (FACScan, Becton Dickinson). Separate
analysis was performed for neutrophils, monocytes, and
lymphocytes by gating according to their characteristic side and
forward light-scatter patterns.4 Gating also permitted
exclusion of free microbubbles from analysis.1
Experiments were performed in duplicate, and the data were displayed as
histograms of green fluorescence in a gated population.
Temporal Characterization of Microbubble-Leukocyte
Interactions
Microscopy was performed to determine the fate of microbubbles
after their attachment to activated leukocytes. Samples (50
µL) were removed 3, 15, and 30 minutes after activated
leukocytes were incubated with microbubbles, placed on a slide, and
observed with an inverted microscope (Axiovert, Carl Zeiss) with an
oil-immersion objective (x100/1.3 numerical aperture). A minimum of 50
cells were identified under transillumination and classified according
to whether they interacted (by attachment or phagocytosis) with
microbubbles.
For transmission electron microscopy (TEM), samples (150 µL) from the leukocyte-microbubble suspensions were placed in an equivalent volume of 0.1 mol/L sodium cacodylate buffer (pH 7.5) containing 2% osmium tetroxide for 30 minutes. They were centrifuged, washed in PBS, fixed in 2% glutaraldehyde/paraformaldehyde, dehydrated in a graded series of acetone, and embedded in epoxy resin. Thin sections were stained with saturated uranyl acetate and lead citrate. Observations were made at final magnification of x6600 with a transmission electron microscope (100CX, Jeol).
Microbubble Responses to US
To evaluate the acoustic activity of phagocytosed microbubbles,
we first performed in vitro studies by using a system that allowed
simultaneous US exposure and light microscopy. A syringe
containing microbubble-leukocyte suspensions was connected to a 4- to
6-cm segment of cellulose tubing with an internal diameter of 200
µm. This tubing was immersed in a water bath secured to the stage of
a light microscope (IV500L, Mikron Instruments) and positioned in the
focal planes of a water immersion objective (SW 100/1.3 numerical
aperture) and a 2.25-MHz, spherically focused US transducer (V306,
Panametrics) oriented perpendicular to each other. The transducer was
interfaced to a square-wave pulse generator (SP-801A, Ritec) to produce
broad-band pulses of 1.5 cycles at the center frequency. Acoustic
pressure measurements were made at the focal plane before each
experiment with a calibrated needle hydrophone (PZT 2422-0200,
Specialty Engineering Associates).
Activated leukocyte and microbubble suspensions were placed in a microinjector (IM-5B, Narishige) that allowed positioning of a single cell or microbubble in the optical field. Free and phagocytosed microbubbles were repeatedly exposed to single pulses of US with peak negative acoustic pressures of -400, -940, or -1600 kPa. Recordings were made with a video camera (Motioncorder 1000, Kodak) interfaced with an S-VHS recorder (AG-1980, Panasonic).
Microbubble diameters were measured off-line with video calipers, and microbubble volume was calculated assuming a spherical geometry. The rate of reduction in microbubble size during US pulsing was determined by fitting a monoexponential function, y=e-ßx+C, to the relation between pulse number and microbubble volume, where y is normalized microbubble volume, ß is the time constant of the decay, and C is a constant.
In Vivo Detection of Phagocytosed Microbubbles
The study protocol was approved by the animal research committee
at the University of Virginia. Six male wild-type C57BL/6 mice (22 to
30 g) were anesthetized with an
intraperitoneal injection of a solution containing
ketamine hydrochloride, xylazine, and atropine. Body
temperature was maintained at 37°C with a heating pad. Both jugular
veins were cannulated for administration of microbubbles and drugs.
Anesthesia was maintained with intravenous
administration of 0.1 mg pentobarbital every 45 minutes as needed. A
cremaster muscle was prepared for intravital microscopy.5
The muscle was exteriorized through a scrotal incision and secured to a
translucent pedestal. A longitudinal incision was made in the muscle,
and the edges were secured to the pedestal. The preparation was
superfused continuously with isothermic bicarbonate-buffered
saline.
Microscopic observations were made with an intravital microscope (Axioskop 2 FS, Carl Zeiss, Inc) with a saline immersion objective (SW 40/0.8 numerical aperture). Epifluorescent imaging was performed with an excitation filter for fluorescein (460 to 500 nm). Video recordings were made with a high-resolution CCD camera (C2400, Hamamatsu Photonics) connected to an S-VHS recorder (S9500, JVC).
Centerline venular red blood cell velocities were measured with a
dual-slit photodiode6 (CircuSoft Instrumentation) and
converted to mean velocities by multiplying by 0.625.7
Shear rates (
w) were determined by means of
the equation
w=2.12(8Vb)/d, where
Vb is the mean blood velocity, d is the vessel
diameter measured off-line with video-calipers, and 2.12 is a
correction factor for the velocity profile.8 Adherent
leukocytes, defined as those not moving for 
30 seconds, were counted
and expressed per venular surface area, calculated from offline
diameter and length measurements.
US was performed with a Sonos 5500 system (Agilent Technologies) with harmonic imaging at transmit and receive frequencies of 1.8 and 3.6 MHz, respectively. The US transducer was placed in a bath containing isothermic bicarbonate-buffered saline surrounding the pedestal and positioned perpendicular to the microscope objective. A mechanical index of 0.9 and a compression of 75% were used. Gain was optimized at the beginning of each experiment and was held constant. The US pulsing interval (PI) was controlled with an internal timer. Video recordings were made with an S-VHS recorder (MD-830, Panasonic).
Inflammation of the cremaster was produced in 3 mice by intrascrotal
injection of 0.5 µg of murine recombinant TNF- (Sigma) in 0.2 mL
saline 2 hours before dissection, and 3 mice served as controls.
Observations were made in venules with diameters between 25 and 40
µm. To assess venular hemodynamics and the degree of
leukocyte adhesion, 3 venules were recorded under transillumination
followed by measurement of centerline blood velocity. Immediately after
intravenous injection of 2x107
fluorescein-labeled microbubbles, 20 separate high-power
fields encompassing the venules were observed over a period of 2
minutes with fluorescent epi-illumination. These observations
were repeated 15 minutes after injection of microbubbles. Because of
the potential of US to destroy microbubbles,9 10 US
examination was not performed at either of these 2 stages.
US imaging was initiated 17 minutes after microbubble injection, when the blood pool concentration of freely circulating microbubbles should be negligible. The video intensity (VI) in the first frame on resumption of US imaging should reflect the total tissue concentration of microbubbles (adhered, phagocytosed, and freely circulating); that of the next frame should reflect microbubbles that have either not been destroyed or that have reentered the region from the circulating blood pool.11 If the microbubble blood pool concentration is low, and if nearly all microbubbles are destroyed by the first US pulse, then the VI should be much lower in the second than in first pulse. Accordingly, on resumption of US imaging, a first set of 5 images was obtained at a PI of 0.5 seconds followed by a second set of 3 images at a PI of 30 seconds. The second set of images was obtained to assess the signal from freely circulating microbubbles, because microbubble replenishment into tissue should be complete 30 seconds after a US pulse.11
US images were analyzed offline as previously described.12 Several precontrast frames (obtained before microbubble injection) were averaged to create a baseline image. This image was then subtracted from each of the images from the first set as well as a similarly averaged image from the second set.
Statistical Analysis
Data are expressed as mean±SD. Nominal comparisons were made by
Fisher’s exact test. Repeated-measures ANOVA was used to compare flow
cytometry data and to determine differences in rate constants of
microbubble decay in vivo. Comparisons of in vivo data were made by
means of a Student’s t test. Differences were considered
significant at P<0.05 (2-sided).
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Results |
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Cell Types Involved in Leukocyte-Microbubble Interactions
On flow cytometry, fluorescein-labeled microbubbles alone were intensely fluorescent, whereas leukocytes had little fluorescence (Figure 1A
). Figure 1B shows examples of histograms obtained after lipid
microbubbles were combined with neutrophils and free microbubbles were
excluded from analysis by gating to light-scatter pattern.
Microbubble attachment to neutrophils was indicated by the appearance
of a fluorescent peak associated with these cells, which was
greater when cells were activated by PMA. After activation, the
proportion of neutrophils interacting with microbubbles increased from
0.15±0.03 to 0.47±0.05 (P<0.05) for lipid microbubbles
and from 0.17±0.04 to 0.85±0.07 for albumin microbubbles
(P<0.05). The proportion of monocytes interacting similarly
increased from 0.16±0.04 to 0.60±0.05 (P<0.05) and from
0.06±0.01 to 0.12±0.02 (P<0.05) for albumin and
lipid microbubbles, respectively.
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Temporal Characterization of Leukocyte-Microbubble
Interactions
The sequence of microbubble-leukocyte interactions over time is
illustrated in Figure 2. Early (3 minutes
after their combination), microbubbles attached to the cell surface of
activated leukocytes, ranging from 1 to 6 microbubbles per
leukocyte. Most of these microbubbles were phagocytosed intact by 15
minutes. By 30 minutes, however, intracellular albumin
microbubbles were degraded into shell and bubble fragments, whereas
lipid microbubbles remained intact. At all time points, the percentage
of cells interacting with either microbubble type was less
(P<0.05) for the lymphocyte-monocyte fraction than for
neutrophils (<10% compared with >50% of cells).
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Confirmation of the intracellular location of microbubbles was made
with TEM (Figure 3), since microbubbles
attached to leukocytes might assume a polar orientation on the cell
surface as the result of buoyancy and therefore appear to be
intracellular on light microscopy. At 30 minutes, albumin
microbubble shells were significantly degraded with adjacent organelle
paucity, consistent with intracellular retention of the
perfluorocarbon gas (Figure 3). In comparison, lipid
microbubbles remained intact at this time point.
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Response of Phagocytosed Microbubbles to US
In the in vitro studies, the mean microbubble diameters before US
exposure were not significantly different for free versus phagocytosed
albumin (3.16±0.64 versus 2.94±0.62 µm) or lipid
(3.42±1.06 versus 3.08±0.82 µm) microbubbles. Figure 4 depicts the effect of repetitive single
pulses of US on the size of single phagocytosed albumin
microbubbles viewed under light microscopy. At a low acoustic pressure
(-400 kPa), each US pulse resulted in minimal change in microbubble
size compared with baseline. At a moderate acoustic pressure (-940
kPa), individual pulses of US resulted in sequential reductions in
microbubble size until destruction was complete. At a high acoustic
pressure (-1600 kPa), albumin microbubbles were destroyed by a
single US pulse. Microbubble destruction at this pressure frequently
caused either distortion of the cell membrane within seconds of
microbubble destruction or immediate rupture of the neutrophil cell
membrane, indicated by collapse of the cell and efflux of its
cytoplasmic granules into the surrounding medium. These effects were
also observed with lipid microbubble destruction at -1600 kPa.
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Figure 5 illustrates the rates of decline
in microbubble volume with repetitive pulses of US at various peak
negative acoustic pressures. Peak negative pressures of -400 kPa had
little effect on phagocytosed microbubbles, regardless of their
composition, but produced a gradual decline in volume of free
microbubbles. At a peak negative pressure of -940 kPa, sequential US
pulses resulted in incremental reductions in microbubble volumes of
both free and phagocytosed microbubbles. At -1600 kPa, a single US
pulse caused destruction of all albumin microbubbles, whereas
lipid microbubble destruction required several pulses. Overall,
increasing the acoustic pressure resulted in a greater
(P<0.05) rate of decline in microbubble size, irrespective
of shell composition or location (free versus intracellular). The rate
of decay in size at each pressure was lower for the intracellular
microbubbles. The only exception was lipid microbubbles exposed to
-1600 kPa, in which the difference in the decay rates for the free and
intracellular microbubbles was marginal (P=0.05). The time
constants of decay at this pressure were not calculated for
albumin microbubbles because of their complete destruction with
the first US pulse.
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) and
phagocytosed (
) albumin and lipid microbubbles at baseline
(BL) and after sequential pulses of US (P1 through
P5) at peak negative acoustic pressures of -400, -940,
and -1600 kPa. Rate constants (ß) of decay of microbubble volume are
shown. *P<0.05 vs free microbubbles.
In the in vivo experiments, the mean venular diameter, blood flow
velocity, and shear rate were similar in control and TNF-–treated
mice (Table). The mean number of
adherent leukocytes was >7-fold higher after TNF- treatment than in
controls (Table), indicating an intense inflammatory
response.
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In control mice, microbubble interactions with the few adherent
leukocytes were uncommon when observations were made both early (0 to 2
minutes) and late (15 to 17 minutes) after their
intravenous injection (Figure 6). In comparison, treatment with TNF-
resulted in a far greater number of microbubble interactions with the
abundant adherent leukocytes at both time points. Microbubbles attached
to the leukocyte surface early after injection, and most appeared to be
phagocytosed by 15 to 17 minutes (Figure 7), at which time freely circulating
microbubbles were only occasionally observed (<1 microbubble
transiting the venules every 10 seconds). Unlike in the in vitro
setting, no obvious disruption of the leukocyte membrane was observed
when phagocytosed microbubbles were insonified.
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Figure 8 illustrates examples of US
images of the cremaster muscles in a control and a TNF-–treated
mouse. In the control animal, the VI increased minimally 17 minutes
after lipid microbubble injection compared with baseline (precontrast)
from the few freely circulating microbubbles passing through the
tissue. In comparison, intense contrast enhancement was seen at this
time in the TNF-–treated mouse, which mostly
represented signal from the phagocytosed/attached
microbubbles retained at the site of inflammation. All microbubbles
were destroyed by the initial pulse of US so that the VI in the next
frame, obtained 0.5 second later, was reduced and resembled that of the
baseline image. The VI increased minimally when the PI was prolonged to
30 seconds, which allowed complete replenishment of the tissue from
circulating microbubbles. The VI in this frame was much less than that
seen in the first frame obtained after microbubble injection in the
TNF-–treated animal, indicating that the signal from the few freely
circulating microbubbles was very low.
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The mean background-subtracted VI from the cremaster muscles of all
mice is shown in Figure 9. In control
mice, the VI was low in the initial frame obtained on resumption of
imaging 17 minutes after injection of albumin or lipid
microbubbles. Because of US-induced microbubble destruction, the VI
decreased slightly on the next frame obtained 0.5 second later. The
mean VI from subsequent images obtained at a PI of 30 seconds, which
represented signal from freely circulating microbubbles,
was not significantly different from the initial frame. The mean
background-subtracted VI on the initial frame on resumption of imaging
17 minutes after microbubble injection was 5- to 6-fold greater
(P<0.01) in TNF-–treated compared with control animals.
The VI on the next frame 0.5 second later was much lower as a result of
microbubble destruction and increased only marginally at a PI of 30
seconds from tissue replenishment by freely circulating microbubbles.
These results indicate that the intense signal noted on resumption of
imaging 17 minutes after microbubble injection was due to phagocytosed
or attached microbubbles retained within the inflamed tissue rather
than those freely circulating in the blood pool.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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We have recently reported that microbubbles attach to activated leukocytes adherent to the venular endothelium after ischemia-reperfusion and TNF-
–induced inflammation.1 The new information
obtained from this study is that (1) neutrophils and monocytes are
responsible for leukocyte-microbubble interactions; (2) after
attachment, microbubbles are phagocytosed intact; and (3) despite
viscoelastic damping, phagocytosed microbubbles remain acoustically
active, thereby permitting their detection by US imaging of inflamed
tissue. These results indicate that with microbubbles, it may be
possible to noninvasively image inflammation in organ systems
accessible to US and also conceivably administer drugs to these
sites.
Characterization of Microbubble-Leukocyte Interactions
We have previously shown that microbubble-leukocyte interactions
are influenced by the microbubble shell composition. Albumin
microbubbles attach to leukocytes through the
ß2-integrin Mac-1 on the surface of the
activated leukocytes, whereas lipid microbubbles attach to
activated leukocytes through opsonization by serum
complement.1 In this study, we found that the cells
involved were primarily neutrophils and monocytes, both of which
express active Mac-1 and other complement receptors on cell
activation.13 14
Although we have observed attachment of microbubbles in vivo to leukocytes adherent to inflamed venular endothelium,1 the consequent fate of these microbubbles was not defined. In this study, we found that these microbubbles were phagocytosed intact within minutes by activated leukocytes. This finding is consistent with the specific role of Mac-1 and complement receptors in immunoglobulin-independent phagocytosis.15
Response of Phagocytosed Microbubbles to US
Currently, the detection of microbubbles in tissue depends on
their nonlinear behavior in a US field as well as microbubble
destruction that occurs when acoustic pressure is sufficiently
high.9 10 As illustrated in this and previous
studies,9 free microbubble destruction can occur with a
single US pulse delivered at a high negative acoustic pressure. At more
moderate acoustic pressures, the destruction is gradual, with
progressive deflation of the microbubbles with each subsequent US
pulse. Microbubble oscillation at moderate US pressures
most likely results in less severe shell defects with partial release
of gas into the surrounding medium or simply in augmented outward
movement of gas during the compressive phase.9
The magnitude of US-mediated microbubble oscillations will depend on several factors, including the composition of the gas and shell as well as the degree of viscous and thermal damping.16 17 It is probable that oscillations of the phagocytosed microbubbles in this study were damped by the viscoelastic properties of the surrounding cellular milieu, resulting in smaller fluctuations in size and less shell damage compared with free microbubbles. As a result, a lower rate of decline in size was found for phagocytosed compared with free microbubbles at moderate peak negative pressures (-400 and -940 kPa).
Because of differences in the material properties of their shells, albumin and lipid microbubbles behaved somewhat differently at the highest acoustic pressure applied in our experiments (-1600 kPa). At this pressure, albumin microbubbles were more susceptible to destruction than lipid microbubbles. Destruction of either occasionally caused disruption of the neutrophil cell membrane, probably caused by the large amplitude of microbubble oscillations at this pressure. Other potential mechanisms for this effect include local shock or thermal effects or membrane damage caused by outward shell fragmentation.9 These detrimental effects on the leukocyte noted in vitro were not reproduced in vivo at acoustic pressures generated by US systems used clinically.
The change in microbubble size on US exposure in vitro indicated that
despite viscoelastic damping, phagocytosed microbubbles were
acoustically active. To determine whether phagocytosed microbubble
signals could be detected in vivo, we imaged the cremaster muscle of
mice in the presence of TNF-–induced inflammation. The numbers of
both the adherent leukocytes and the microbubbles interacting with them
increased by 7-fold in TNF-–treated animals. US imaging
initiated >15 minutes after microbubble injection (which allowed
microbubble concentration in the blood pool to become negligible)
revealed bright opacification of the cremaster on the initial frame
only in the setting of inflammation. The assumption that this
opacification resulted from microbubbles associated with adherent
leukocytes was supported by the abundant phagocytosed microbubbles seen
by intravital microscopy performed just before initiation of US. Only a
few freely circulating microbubbles were seen and were responsible for
a minimal change in VI at a PI of 30 seconds compared with
baseline.
In conclusion, we have demonstrated that microbubbles are rapidly phagocytosed intact by activated neutrophils and monocytes and can persist intracellularly for almost half an hour before they are degraded. In this period, microbubbles remain acoustically active despite viscoelastic damping caused by the cellular milieu, thereby allowing their detection on US imaging. These findings indicate that contrast-enhanced US may provide a useful means for the noninvasive assessment of inflammation and to follow the response to treatment. Future studies are being directed at optimizing imaging protocols for detection of phagocytosed microbubbles and development of microbubbles with even greater avidity for leukocytes.
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Acknowledgments |
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This study was supported in part by grants (HL-03810 [Dr Lindner], HL-54136 and HL-64381 [Dr Ley], CA-76062 [Dr Ferrara], and HL-48890 [Dr Kaul]) from the National Institutes of Health, Bethesda, Md. The authors are grateful to Alexander L. Klibanov, PhD, for preparation of MP1950 and to Margaretta Allietta, BS, for her assistance with the electron microscopy.
Received October 11, 1999; revision received February 14, 2000; accepted February 29, 2000.
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 P. S. Wild, B. Funke, T. Geisler, A. Abushi, and R. J. Zotz Fragment reconstruction of coronary arteries using transesophageal echocardiography for coronary diagnostics Eur J Echocardiogr, November 1, 2008; 9(6): 796 - 802. [Abstract] [Full Text] [PDF]
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 B. A. Kaufmann, C. Lewis, A. Xie, A. Mirza-Mohd, and J. R. Lindner Detection of recent myocardial ischaemia by molecular imaging of P-selectin with targeted contrast echocardiography Eur. Heart J., August 2, 2007; 28(16): 2011 - 2017. [Abstract] [Full Text] [PDF]
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P.A Dijkmans, L.J.M Juffermans, R.J.P Musters, A van Wamel, F.J ten Cate, W van Gilst, C.A Visser, N de Jong, and O Kamp Microbubbles and ultrasound: from diagnosis to therapy Eur J Echocardiogr, August 1, 2004; 5(4): 245 - 246. [Abstract] [Full Text] [PDF]
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I. Kondo, K. Ohmori, A. Oshita, H. Takeuchi, J. Yoshida, K. Shinomiya, S. Fuke, T. Suzuki, K. Mizushige, and M. Kohno Leukocyte-Targeted Myocardial Contrast Echocardiography Can Assess the Degree of Acute Allograft Rejection in a Rat Cardiac Transplantation Model Circulation, March 2, 2004; 109(8): 1056 - 1061. [Abstract] [Full Text] [PDF]
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H. Leong-Poi, J. Christiansen, A. L. Klibanov, S. Kaul, and J. R. Lindner Noninvasive Assessment of Angiogenesis by Ultrasound and Microbubbles Targeted to {alpha}v-Integrins Circulation, January 28, 2003; 107(3): 455 - 460. [Abstract] [Full Text] [PDF]
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Y. Kono, G. C. Steinbach, T. Peterson, G. W. Schmid-Schonbein, and R. F. Mattrey Mechanism of Parenchymal Enhancement of the Liver with a Microbubble-based US Contrast Medium: An Intravital Microscopy Study in Rats Radiology, July 1, 2002; 224(1): 253 - 257. [Abstract] [Full Text]
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J. R. Lindner, J. Song, J. Christiansen, A. L. Klibanov, F. Xu, and K. Ley Ultrasound Assessment of Inflammation and Renal Tissue Injury With Microbubbles Targeted to P-Selectin Circulation, October 23, 2001; 104(17): 2107 - 2112. [Abstract] [Full Text] [PDF]
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J. R. Lindner, J. Song, F. Xu, A. L. Klibanov, K. Singbartl, K. Ley, and S. Kaul Noninvasive Ultrasound Imaging of Inflammation Using Microbubbles Targeted to Activated Leukocytes Circulation, November 28, 2000; 102(22): 2745 - 2750. [Abstract] [Full Text] [PDF]
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J. P. Christiansen, H. Leong-Poi, A. L. Klibanov, S. Kaul, and J. R. Lindner Noninvasive Imaging of Myocardial Reperfusion Injury Using Leukocyte-Targeted Contrast Echocardiography Circulation, April 16, 2002; 105(15): 1764 - 1767. [Abstract] [Full Text] [PDF]
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