| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Experimental Studies |
1 From the Departments of Radiology (D.L.M., C.D.), Internal Medicine (Cardiology) (P.L., W.F.A.), Pathology (D.G.), and Cell and Developmental Biology (C.A.E.), University of Michigan Medical Center, 3315 Kresge III, 200 Zina Pitcher Pl, Ann Arbor, MI 48109-0553. From the 2004 RSNA Annual Meeting. Received August 25, 2004; revision requested October 29; revision received November 19; accepted December 20. Supported by U.S. Public Health Service grant EB00338, awarded by the National Institutes of Health, Department of Health and Human Services. Address correspondence to D.L.M. (e-mail: douglm{at}umich.edu).
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
MATERIALS AND METHODS: All animal procedures were approved by the university committee for the use and care of animals. Myocardial contrast echocardiography with 1:4 electrocardiographic (ECG) triggering was performed at 1.5 MHz in 61 anesthetized rats. Evans blue (EB) dye was injected as the vital stain for cardiomyocyte injury. At the start of myocardial contrast echocardiography, which lasted 10 minutes, perflutren lipid microsphere–based contrast material was infused through the tail vein for 5 minutes. Premature heartbeats were counted from the ECG record. The numbers of EB-stained cells counted on sections of heart specimens obtained 24 hours after myocardial contrast echocardiography and then either fresh frozen or embedded in paraffin were determined by using fluorescence microscopy. Results were compared statistically by using t tests and Mann-Whitney rank sum tests.
RESULTS: EB-stained cells were concentrated in the anterior region of the myocardium. In the paraffin-embedded specimens, EB-stained cells were often accompanied by but largely separate from areas of inflammatory cell infiltration. At end-systolic triggering with a 50 µL/kg dose of microsphere contrast material, the EB-stained cell count increased with increasing peak rarefactional pressure amplitude, with significantly increased cell counts at 1.6 MPa (P < .02) and 2.0 MPa (P < .005) relative to the cell counts at sham myocardial contrast echocardiography. Premature heartbeats had a similar exposure-response relationship; however, number of premature heartbeats and EB-stained cell count did not appear to be directly related (coefficient of determination r2 = 0.03). The EB-stained cell counts at end-diastolic triggering were not significantly different from those at end-systolic triggering (P > .1). EB-stained cell counts increased with increasing contrast material dose, from 10 to 50 µL/kg, at 2.0 MPa.
CONCLUSION: Cardiomyocyte injury was induced by the interaction of ultrasound pulses with contrast agent microbubbles during myocardial contrast echocardiography in rats, and the numbers of injured cells increased with increasing contrast agent dose and ultrasound exposure.
© RSNA, 2005
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
The interaction between ultrasound pulses and the contrast agent microbubbles is a form of acoustic cavitation, which is an important mechanism of the nonthermal bioeffects of US (3). Bioeffects such as capillary rupture depend specifically on the rarefaction portion of ultrasound pulses (4). Certain types of bioeffects appear to be robust enough to facilitate therapeutic applications performed with existing or specifically designed agents and diagnostic US platforms. These applications include drug delivery and gene therapy (5,6). The bioeffects and therapeutic applications of US related to the contrast agents used have been the subject of several reviews (3,7–9).
Myocardial contrast material–enhanced echocardiography (ie, myocardial contrast echocardiography) performed by using diagnostic US scanners has several potential bioeffects, including premature ventricular contractions (10) without troponin I elevation (11) in humans, microvascular leakage in isolated rabbit hearts (12), mild troponin T elevation without pathohistologic damage after rat heart scanning (13), and microvascular leakage combined with premature heartbeats and surface petechiae in rat hearts (14). The microvascular leakages induced during myocardial contrast echocardiography with different contrast agents have been similar when they were compared according to the number of microbubbles in the doses of each agent (15). The microvascular leakage persists for about 20 minutes after cessation of myocardial contrast echocardiography (16) and is the basis of possible drug delivery applications.
High-pressure-amplitude US (above the diagnostic range) has been shown to cause arrhythmias and pathohistologic myocardial degeneration in rat hearts (17). Histologically identified areas of inflammatory cell infiltration that presumably originated from petechiae reportedly have been depicted at diagnostic US performed with very large (140 times the recommended diagnostic dose) bolus doses of a perflutren protein-type A microsphere–based contrast agent (18).
Research on how diagnostic ultrasound exposure and contrast agent dose influence the extent of cardiomyocyte injury and on the relationship between specific cardiomyocyte injury and other phenomena such as premature heartbeats and inflammatory cell infiltration is warranted. The dose-response relationships between microvascular leakage and different contrast agents have been very different when the same volume doses of the agents were administered but similar when doses with equal microbubble concentrations were administered (15). Thus, cardiomyocyte injury might occur with clinical doses of advanced contrast agents that have relatively high concentrations of microbubbles. Premature heartbeats have not correlated with microvascular leakage, and the two effects have had different exposure-response trends (14,15). Hypothetically, cardiomyocyte injury may be more closely related to premature heartbeats than is microvascular leakage, because both effects (cardiomyocyte injury and premature heartbeats) directly involve the cardiomyocytes. If premature heartbeats were directly correlated with cardiomyocyte injury, then the simple observation of premature heartbeats during myocardial contrast echocardiography would represent a noninvasive indicator of myocardial injury.
The origin and relationships of these potential adverse effects of myocardial contrast echocardiography need to be elucidated to ensure both the safe use of this diagnostic imaging method and a clear separation between diagnostic and therapeutic applications. Thus, the goal of this research was to identify the specific cardiomyocyte injury induced by myocardial contrast echocardiography in rats and to determine the influences of contrast agent dose and ultrasound exposure on this injury.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
US Contrast Agent
We prepared a perflutren lipid microsphere–based contrast agent (Definity; Bristol-Myers Squibb Medical Imaging, North Billerica, Mass) according to the manufacturer's instructions by agitating room-temperature vials of the material for 45 seconds in the vial-mixing machine supplied with the medium. A fresh vial was used each day. This microsphere agent contains a maximum of 12 x 109 stabilized microbubbles per milliliter of a 1.1–3.3-µm microsphere mean diameter. The microbubbles are filled with octafluoropropane and stabilized by a lipid coating. Before being used, the agent was first diluted with sterile saline in a 5-mL syringe, which was then placed in a syringe pump (model 11plus; Harvard Apparatus, Holliston, Mass). We adjusted the dose of the agent by varying the initial dilution; this yielded stock doses of 10, 20, or 50 µL/kg that were administered at 2, 4, or 10 (µL · kg–1)/min, respectively. A 30-cm extension tube was preloaded with diluted contrast agent and connected between the syringe and the tail vein cannula. For infusion, the volume flow rate was set for delivery of 500 (µL · kg–1)/min of diluted agent for 5 minutes.
US Examinations
A Vingmed System V (GE Medical Systems, Cincinnati, Ohio) with a cardiac phased-array probe (FPA2.5; GE Medical Systems) was used for diagnostic US and ultrasound exposure. Two authors (P.L. and D.L.M., with 11 and 2 years of experience in research echocardiography, respectively) clamped the probe in the water bath and used it to acquire a left ventricular short-axis view of the rat heart, which was 4–5 cm from the face of the transducer. This US scanning setup enabled essentially free-field exposure conditions (ie, without multiple echoes from reflecting surfaces) and a focal depth (5 cm) similar to that achieved in human subject scanning. We obtained an initial US scan at 3.6 MHz to clearly visualize the left ventricle; then we switched the system to 1.5 MHz and a 60-Hz frame rate, similar to the parameters used for adult human scanning.
For myocardial contrast echocardiography, frames were triggered intermittently from each fourth beat at electrocardiography. During each scan acquisition, the maximum ultrasound pulse had a peak rarefactional pressure amplitude (PRPA) of 2.3 MPa at the maximum 0-dB power setting and a duration of 1.45 µsec; these parameters were determined in the water bath in the absence of the rat (14). After we made adjustments for the minimum estimated attenuation of 12% (–1.2 dB) through the rat chest wall, the in situ PRPA was 2.0 MPa at the 0-dB power setting, which corresponds to an equivalent mechanical index of 1.6 (ie, the in situ PRPA divided by the square root of the frequency). We adjusted the ultrasound pulse PRPA according to the power level setting, and the measured in situ PRPA was the primary ultrasound exposure parameter. The ultrasound field that reached the myocardium was not uniform. The field decreased with distance from the center of the scanning plane, with a –6 dB thickness of 4.6 mm. Furthermore, the reported maximum PRPA could also be attenuated by the sternum, lungs, ribs, or contrast agent (particularly the contrast agent within the left ventricular cavity). The sternum and lungs were avoided as much as possible, but the ribs normally caused shadowing within the myocardium. The ribs were about 1 mm in diameter and spaced 4–7 mm apart. The mean attenuation directly behind the ribs was a factor of 0.68 ± 0.06 (standard deviation) (or –3.3 dB, the average for six ribs).
The nonuniform PRPA resulted in a nonuniform distribution of bioeffects within the scanning plane and in reduced overall effects (relative to the uniform exposure of the entire heart to the maximum PRPA). For myocardial contrast echocardiography, the infusion was continued for 5 minutes and followed by an additional 5 minutes of triggered scanning. For sham myocardial contrast echocardiography, the two conditions of ultrasound exposure alone and contrast agent alone were combined (ie, echocardiography for 10 minutes followed by infusion of the contrast agent with the US scanning probe aimed away from the rat). The echocardiographic images, including electrocardiographic traces, were recorded for later analysis of the premature heartbeats.
Measured End Points
Cellular injury in the myocardium can be detected by using several methods. The nuclei of dead cells can be identified with propidium iodide staining, but this stain is not specific to cardiomyocytes (19). Antimyosin antibody labels permeabilized cardiomyocytes, but such labeling can be nonspecific and requires subsequent immunofluorescent staining of the antibody label (20). For the current research, Evans blue (EB) dye (Sigma-Aldrich, St Louis, Mo) staining of cardiomyocytes was used to detect cardiomyocyte injury on the basis of its use for myocyte staining, for example, in muscular dystrophy research (21). EB dye rapidly binds to albumin in the blood and is excluded by viable cell membranes. Tests of the postexposure time to observe the EB staining of cardiomyocytes indicated that 24-hour postexposure observation allowed much closer identification of stained cells than 4- or 48-hour postexposure observation, and only 24-hour postexposure observations were included in this study. Cardiomyocyte injury was assessed by observing the red fluorescence of EB-stained cells 24 hours after myocardial contrast echocardiography. EB dye in sterile saline (50 mg/mL) was injected at 100 mg/kg 5 minutes before myocardial contrast echocardiography.
The day after myocardial contrast echocardiography, the rats were sacrificed according to guidelines of the university committee for the use and care of animals. Their hearts were removed (by P.L. and C.D.), perfused with heparin-saline solution or neutral buffered formalin, and sliced to obtain specimens of the US scanning plane, which was often indicated by blue coloration. Fixing the heart specimens (from seven rats) in neutral buffered formalin was valuable for preserving the morphologic features of the tissue. However, the EB-stained cells were normally counted (or scored) on fresh-frozen sections (from 54 rats) to avoid loss of stain, which occurred during the processing of formalin-fixed specimens for paraffin embedding and staining of sections.
To prepare formalin-fixed tissue, the specimens were stored in neutral buffered formalin. Examination of the anterior surface of a whole heart with a stereomicroscope (model MZ FL III; Leica Microscopy Systems, Deerfield, Ill) with fluorescence attachments revealed that red fluorescent cells could be discerned in the tissue as bright red streaks against a darker background. Confocal microscopy (with a model LSM 510 microscope; Carl Zeiss MicroImaging, Thornwood, NY) was performed (by C.A.E.) for visualization of the spatial orientation of the cells, with confocal optics greatly reducing the background. Three heart specimens were prepared in 20-µm-thick fixed-frozen slices, with the nuclei counterstained with 4',6-diamidine-2-phenylindole (Vectashield Mounting Medium with DAPI; Vector Laboratories, Burlingame, Calif). In addition, four formalin-fixed specimens were paraffin embedded, sectioned, and stained with hematoxylin; the normal eosin component of histologic stain, which is fluorescent, was omitted. These histologic slides were prepared at the Research Histology and Immunoperoxidase Laboratory (University of Michigan Comprehensive Cancer Center Tissue Core, Ann Arbor, Mich). These sections were placed in a position estimated to be the center of the scanning plane (but not rigorously determined to be at the position of maximum effect). Areas of interest were measured on photomicrographs by using image analysis software (SigmaScan Pro 5; SPSS Science, Chicago, Ill) and calculated as a percentage of the total tissue area on the photomicrographs.
To prepare fresh-frozen heart sections, the specimens obtained from 54 rats were embedded in medium for frozen tissue specimens (Tissue-Tek; Sakura Finetek, Torrance, Calif) and frozen at –80°C. The scoring plan was developed (by D.L.M. and D.G., with 2 and 20 years of research cardiovascular pathology experience, respectively) to determine the maximal number of stained cells. Frozen sections were made (by P.L. or C.D.) by using a cryostat microtome (model CM 1800; Leica Microsystems). For microscopic fluorescence examination, a 10-µm section was mounted on a microscope slide. The sections were spaced every 0.2 mm into the sample until a maximal number of EB-stained (fluorescent) cells were counted, with declining counts in sections obtained deeper in the sample. An author (D.L.M.) carefully scored the section from each heart that was estimated to have the maximal number of stained cells by counting the individual EB-stained cells and visually estimating the number of cells in the EB-stained groups of cells in the entire section. Staining was evident as faint blue regions for bright-field illumination but was much more easily discernible by using epifluorescent illumination for EB dye fluorescence with 515–560-nm-band-pass excitation and 590-nm-long-pass suppression filters (model DMRB fluorescence microscope; Leica Microscopy Systems). Finally, premature heartbeats were counted (by P.L. and C.D.) from the electrocardiogram recorded on videotape.
Experimental Plan
Experiments were conducted in three series of rat exposures to ultrasound, which were performed at different times but with essentially identical methods. These three series were (a) observation of the spatial distribution of EB-stained cells, (b) determination of the PRPA exposure response for EB-stained cell counts, and (c) determination of the contrast agent dose response for EB-stained cell counts. The spatial distribution observations were performed to examine the spatial distribution of the fluorescent cells. For this purpose, thick fixed slices were examined with confocal microscopy, with one sham myocardial contrast echocardiographic exposure, one exposure at 2.0 MPa with a 20 µL/kg microsphere contrast agent dose, and one exposure at 2.0 MPa with a 50 µL/kg dose. In addition, four thin sections were examined with transmitted light microscopy plus fluorescence microscopy, with all of the sampled regions having been imaged at myocardial contrast echocardiography with a 50 µL/kg contrast agent dose and 2.0 MPa. These methods allowed separate observations of the fluorescent body of the injured cells and of the cell nuclei of all the cells, and these observations permitted partial identification of the cells as myocytes, infiltrating inflammatory cells, or other cells.
The PRPA exposure-response series was conducted to determine the influence of PRPA on cardiomyocyte injury with use of fresh-frozen sections. This series included end-systole triggering with a 50 µL/kg contrast agent dose for the sham myocardial contrast echocardiography examinations (five specimens), myocardial contrast echocardiography at 0.6 MPa (–12 dB power setting) (four specimens), myocardial contrast echocardiography at 0.74 MPa (–10 dB) (five specimens), myocardial contrast echocardiography at 1.1 MPa (–6 dB) (five specimens), myocardial contrast echocardiography at 1.6 MPa (–3 dB) (five specimens), and myocardial contrast echocardiography at 2.0 MPa (0 dB) (six specimens). In addition, end-diastole triggering was used for myocardial contrast echocardiography at 2.0 MPa (0 dB) (five specimens).
The contrast agent dose-response series was conducted to determine the influence of contrast agent dose on the degree of cardiomyocyte injury in rat hearts with use of fresh-frozen sections. This series included end-systole triggering at 2.0 MPa for myocardial contrast echocardiography (six specimens) with a 10 µL/kg contrast agent dose, myocardial contrast echocardiography with a 20 µL/kg dose (seven specimens), and myocardial contrast echocardiography with a 50 µL/kg dose (six specimens). The five sham myocardial contrast echocardiography samples from the PRPA exposure-response series of experiments were also included in this series, and the identical conditions of myocardial contrast echocardiography performed at 2.0 MPa with a 50 µL/kg contrast agent dose applied in both these series were pooled to yield a total of 12 tests performed by using this condition.
Statistical Analyses
Numerical results are either presented as means ± standard deviations or plotted as means with standard error bars. Groups of four to seven replicate tests were sufficient to demonstrate the large difference between the sham myocardial contrast echocardiography and myocardial contrast echocardiography exposure conditions. For statistical analyses, which were performed by using computer software (SigmaStat 3.1; Systat Software, Point Richmond, Calif), Student t tests or Mann-Whitney rank sum tests were used, as appropriate, to compare the means of the measured parameters, with statistical significance assumed at P < .05. Linear or nonlinear regression analysis was performed, as appropriate, to aid in the interpretation of results.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
|
|
Response to PRPA Exposure
Compared with sections from formalin-fixed, paraffin-embedded specimens, the sections from fresh-frozen specimens were less morphologically intact but had a smaller loss of stain and thus yielded bright fluorescence for rapid scoring. A photomicrograph of a fresh-frozen section with EB-stained cells is shown in Figure 1, B. The fluorescent background of the interstitium and the fluorescent features of structures such as arteriole walls were excluded in the scoring of these sections. The number of affected cardiomyocytes—that is, the number of EB-stained cells—was determined over entire slices. The fluorescent cells were much more clearly discernible on the sections from fresh-frozen specimens than on the sections from paraffin-embedded specimens; however, areas of inflammatory cell infiltration were not clearly defined on the sections from frozen specimens.
In the PRPA exposure-response series of tests performed to assess the effects of different PRPAs, the number of EB-stained cells in the samples exposed to most of the PRPAs at myocardial contrast echocardiography was not significantly different from the number of cells in the samples exposed to sham myocardial contrast echocardiography, as shown in Figure 3, A. Relative to the cell counts at sham myocardial contrast echocardiography, the cell counts at 1.6 MPa (P < .02) and 2.0 MPa (P < .002)—but not those at 1.1 MPa (P > .05)—were significantly increased. The numbers of premature heartbeats that occurred during myocardial contrast echocardiography are shown in Figure 3, B. The two effects (EB-stained cell count and number of premature heartbeats) appeared to have similar variations with the PRPA, and both effects were significant at the two highest PRPAs relative to these effects at the sham myocardial contrast echocardiographic examinations. These findings appear to support the hypothesis that a relationship exists between these effects. However, regression analysis of the 12 data pairs obtained by using the highest PRPA indicated no direct correlation (coefficient of determination r2 = 0.03) between number of premature heartbeats and EB-stained cell count. Therefore, number of premature heartbeats was not directly related to degree of cell injury.
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
At end-systolic triggering with a 50 µL/kg contrast agent dose, the number of EB-stained cells increased with increasing PRPA, with significantly increased numbers at 1.6 MPa (P < .02) and 2.0 MPa (P < .005) relative to the counts at sham myocardial contrast echocardiography. There was a similar relationship between the number of premature heartbeats and PRPA exposure; however, number of premature heartbeats and EB-stained cell count were not directly correlated according to the results of regression analysis. The lack of a direct correlation between these effects indicated that there probably was no causal relationship (eg, cell injury causing a premature heartbeat). Thus, the observation of a premature heartbeat in a human subject during myocardial contrast echocardiography does not necessarily imply irreversible cardiomyocyte injury. The EB-stained cell counts at end-diastolic triggering were not significantly different from those at end-systolic triggering. The numbers of premature heartbeats at end-diastolic triggering were greatly reduced, however. This large reduction in the number of premature heartbeats was expected owing to the depolarized condition of the cells at end diastole (10,14).
The number of EB-stained cells increased with increasing contrast agent dose, from 10 to 50 µL/kg, at 2.0 MPa, and appeared to fit a simple exponential model. In this model, the number of EB-stained cells was approximately proportional to the contrast agent dose at low doses and leveled off at high doses. This simple model has also been applied in the observation of surface petechiae (15).
The numbers of EB-stained cells over entire slices (mean number, 192 ± 152 at 2.0 MPa at the recommended contrast agent dose of 10 µL/kg) were small relative to the number of cardiomyocytes over entire sections. However, in the strongly affected regions in the anterior part of the myocardium, the injury involved a substantial fraction of small photographed areas (eg, 17.1% on the photomicrographs of hematoxylin-stained paraffin slices imaged with myocardial contrast echocardiography at 2.0 MPa and a 50 µL/kg contrast agent dose). The reason for the high concentration of injured cells in portions of the anterior region of the left ventricular wall was probably the nonuniform ultrasound field caused by ribs, lung, or contrast agent in the left ventricle, which shadowed the posterior region of the left ventricular wall (see Materials and Methods). The areas of injury were sometimes characterized by a lengthwise linear arrangement of injured cells. The EB-stained cells often extended beyond associated areas of inflammatory cell infiltration but were also seen separately. The exact causes of these different bioeffects are unknown.
Areas of inflammatory cell infiltration likely arise from the sites of petechial hemorrhage seen immediately after myocardial contrast echocardiography (14,15). The EB-stained cells appear to represent additional cell injury, possibly occurring after the petechiae, in association with areas of inflammatory cell infiltration. Several processes that may be involved in the delayed injury and grouping of EB-stained cells have been identified in cardiac injury research. These processes include transmission of injury in cardiomyocytes through gap junctions to adjacent cells (22), initiation of relatively slow cell death by means of apoptosis (23), and cellular effects of neutrophil activity on nearby cells (24).
Practical applications: Commercially available US contrast agents such as Definity have been tested for their clinical safety and effectiveness in diagnostic applications. Basic research has revealed that the potential for cardiomyocyte injury exists with use of US contrast agents. Use of contrast agents in conventional and magnetic resonance imaging examinations has often been associated with side effects (25,26). However, US contrast agents appear to be unique in that they have effects that depend on the interaction between the ultrasound pulses and the stabilized microbubbles (rather than pharmacologic effects in the absence of the imaging modality). This factor facilitates a reduction or the prevention of the potential effects because ultrasound exposure parameters are altered. High-mechanical-index myocardial contrast echocardiography with end-systole triggering has been considered for use in myocardial perfusion imaging (27–29). However, the Definity package insert includes general precautions that the safety of activated Definity has not been established for mechanical index values greater than 0.8 or end-systole triggering. In this study, cardiomyocyte injury was not significant at 1.1-MPa end-systole triggering, which corresponds to an equivalent mechanical index of 0.9. Thus, the potential adverse effects of the cardiomyocyte injury during myocardial contrast echocardiography described herein could be averted by following the general precautions noted in the package insert.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Abbreviations: EB = Evans blue • PRPA = peak rarefactional pressure amplitude
Authors stated no financial relationship to disclose.
Author contributions: Guarantor of integrity of entire study, D.L.M.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; approval of final version of submitted manuscript, all authors; literature research, D.L.M., W.F.A.; experimental studies, D.L.M., P.L., C.D., C.A.E.; statistical analysis, D.L.M.; and manuscript editing, D.L.M., P.L., D.G., C.A.E.
|
References |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  J. C. Chappell, J. Song, A. L. Klibanov, and R. J. Price Ultrasonic Microbubble Destruction Stimulates Therapeutic Arteriogenesis Via the CD18-Dependent Recruitment of Bone Marrow-Derived Cells Arterioscler. Thromb. Vasc. Biol., June 1, 2008; 28(6): 1117 - 1122. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Vancraeynest, J. Kefer, C. Hanet, C. Fillee, C. Beauloye, A. Pasquet, B. L. Gerber, M. Philippe, and J.-L. J. Vanoverschelde Release of cardiac bio-markers during high mechanical index contrast-enhanced echocardiography in humans Eur. Heart J., May 2, 2007; 28(10): 1236 - 1241. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. L. Miller, E. M. Driscoll, C. Dou, W. F. Armstrong, and B. R. Lucchesi Microvascular Permeabilization and Cardiomyocyte Injury Provoked by Myocardial Contrast Echocardiography in a Canine Model J. Am. Coll. Cardiol., April 4, 2006; 47(7): 1464 - 1468. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| RADIOLOGY | RADIOGRAPHICS | RSNA JOURNALS ONLINE |
