Chunyan Dou

DNA transfer and cell killing in epidermoid cells by diagnostic ultrasound activation of contrast agent gas bodies in vitro

DNA transfer by sonoporation and cell killing in monolayer cells were examined by contrast-aided low-power diagnostic ultrasound (US). Culture chambers with epidermoid cell monolayers were scanned at about 1 mm/s with a 1.5-MHz scan head aimed upward at the chamber in a 37 degrees C water bath. For DNA transfer tests, plasmids coding for green fluorescent protein (GFP) were added to the medium, and GFP expression was assessed by flow cytometry after 2 days. In separate tests, cell killing was determined immediately after treatment. GFP-positive cell counts were 0.4% (0.7% SD) for shams and 3.7% (1.2% SD) of cells for exposure at 2.3 MPa with 2% Optison contrast agent. The fraction of dead cells was 3.4% (1.7% SD) in shams and 28.6% (6.3% SD) in exposed chambers. Both effects increased for increasing Optison concentration and increasing peak rarefactional pressure amplitude. Contrast-aided diagnostic US has a potential therapeutic application for gene transfer, but a trade-off appears to exist with cell killing.

Induction of apoptosis in sonoporation and ultrasonic gene transfer.

The role of apoptosis in sonoporation and ultrasound-enhanced gene transfection of cell suspensions was examined in vitro. Suspensions of HL-60 and of CHO-K1 cells were exposed to 2.25-MHz continuous ultrasound for 1 min in a 60-rpm rotating-tube exposure system, with ultrasound contrast media added to ensure nucleation of cavitation. Cell necrosis was measured by trypan blue dye exclusion (using a hemacytometer) and by propidium iodide nuclear staining (using flow cytometry). Apoptosis was detected by the annexin V method with Alexa Fluor 350 as the fluorescent label, and confirmed by Hoechst 33342 nuclear staining. Sonoporation cell loading was assessed by uptake of large fluorescent-dextran molecules from the medium. Transfection was demonstrated by expression of green fluorescent protein (GFP) from plasmids transferred into the cells by the treatment. Cell scoring was performed by flow cytometry, with necrotic cell events excluded. For HL-60 cells at 0.4 MPa, cell loading and transfection was significantly increased relative to shams at 2, 6 and 24 h post exposure, peaking at 19.0 +/- 5.5% and 9.6 +/- 4.2% of non-necrotic cells, respectively, at 6 h. However, about one third of the treatment-positive cells were identified as apoptotic. The cell loading and gene transfer effects increased for increasing peak rarefactional pressure amplitude, reaching 24.4 +/- 7.7% and 12.7 +/- 5.1% of non-necrotic cells, respectively, for 0.6-MPa exposure. However, the lethal cellular injury caused by cavitation in the rotating tube system reduced the overall apparent efficacy of cell loading and gene transfer to 5.1 +/- 2.1% and 2.1 +/- 0.9%, respectively, after accounting for necrosis and apoptosis. Similar tests with CHO cells showed increased sonoporation but mostly cell death by necrosis, rather than apoptosis. The induction of apoptosis by cavitation treatments should be considered as a possible confounding factor, in addition to necrosis, in sonoporation and ultrasonic gene transfer research.

Frequency Dependence of Kidney Injury Induced by Contrast-Aided Diagnostic Ultrasound in Rats

This study was performed to examine the frequency dependence of glomerular capillary hemorrhage (GCH) induced by contrast-aided diagnostic ultrasound (DUS) in rats. Diagnostic ultrasound scanners were used for exposure at 3.2, 5.0 and 7.4 MHz, and previously published data at 1.5 and 2.5 MHz was also included. A laboratory exposure system was used to simulate DUS exposure at 1.0, 1.5, 2.25, 3.5, 5.0 and 7.5 MHz, with higher peak rarefactional pressure amplitudes (PRPAs) than were available from our DUS systems. The right kidneys of rats mounted in a water bath were exposed to intermittent image pulse sequences at 1 s intervals during infusion of diluted ultrasound contrast agent. The percentage of GCH was zero for low PRPAs, and then rapidly increased with increasing PRPAs above an apparent threshold, p(t). The values of p(t) were approximately proportional to the ultrasound frequency, f, such that p(t) /f was approximately 0.5 MPa/MHz for DUS and 0.6 MPa/MHz for laboratory system exposures. The increasing thresholds with increasing frequency limited the GCH effect for contrast-aided DUS, and no GCH was seen for DUS at 5.0 or 7.4 MHz for the highest available PRPAs.

Are ECG Premature Complexes Induced by Ultrasonic Cavitation Electrophysiological Responses to Irreversible Cardiomyocyte Injury

The objective of this study was to explore the relationship between premature complexes (PCs) in the electrocardiogram (ECG) and lethal injury of cardiomyocytes induced by ultrasound exposure of the heart with contrast-agent gas bodies in the circulation. Anesthetized rats were exposed in a heated water bath to 1.55 MHz focused ultrasound with bursts triggered at end systole during contrast agent infusion. PCs were detected in ECG recordings and cardiomyocyte necrosis was scored by identifying Evans blue-stained cells in multiple frozen sections. With 0.1 μL/kg/min infusion of contrast agent for 5 min, both effects increased strongly for 2-ms bursts with increasing peak rarefactional pressure amplitude >1 MPa. At 8 MPa, statistically significant effects were found even for no agent infusion relative to sham tests. For 2-ms bursts at 2 MPa, the highly significant bioeffects seen for 10-, 1- and 0.1-μL/kg/min infusion became marginally significant for 0.01 μL/kg/min, which indicated a lower probability of cavitation nucleation. Burst duration variation from 0.2-20 ms produced no substantial trends in the results. Overall, the two effects were well correlated (r(2) = 0.88). The PCs occurring during contrast-enhanced ultrasound therefore appear to be electrophysiological responses to irreversible cardiomyocyte injury induced by ultrasonic cavitation.

Optimization of Ultrasound Parameters of Myocardial Cavitation Microlesions for Therapeutic Application

Intermittent high intensity ultrasound scanning with contrast microbubbles can induce scattered cavitation microlesions in the myocardium, which may be of value for tissue reduction therapy. Anesthetized rats were treated in a heated water bath with 1.5 MHz focused ultrasound pulses, guided by an 8 MHz imaging transducer. The relative efficacy with 2 or 4 MPa pulses, 1:4 or 1:8 trigger intervals and 5 or 10 cycle pulses was explored in six groups. Electrocardiogram premature complexes (PCs) induced by the triggered pulse bursts were counted, and Evans blue stained cardiomyocyte scores (SCSs) were obtained. The increase from 2 to 4 MPa produced significant increases in PCs and SCSs and eliminated an anticipated decline in the rate of PC induction with time, which might hinder therapeutic efficacy. Increased intervals and pulse durations did not yield significant increases in the effects. The results suggest that cavitation microlesion production can be refined and potentially lead to a clinically robust therapeutic method.

Glomerular Capillary Hemorrhage Induced in Rats by Diagnostic Ultrasound with Gas–Body Contrast Agent Produces Intratubular Obstruction

Glomerular capillary hemorrhage (GCH) induced by ultrasonic cavitation during diagnostic imaging represents a unique contrast agent-related nephron injury. Consequences of GCH during 1.5-MHz diagnostic ultrasound with contrast agent were examined by histologic methods in rats. Definity was infused at 10 microl/kg/min for 5 min at the start of 8 min of intermittent image-exposure, with 2.3 MPa in situ peak rarefactional pressure amplitude. Kidney samples were taken for histology at 5 min, 30 min, 4 h, 2 d, 1 week and 4 weeks post exposure. In addition, samples were taken at 4 h from groups treated with heparin or aminocaproic acid. GCH was found in 61% of glomeruli in the center of the scan plane 5 min after exposure, which declined (p < 0.05) to 36.3% after 4 h. The width of Bowmans space was significantly increased for glomeruli with GCH relative to glomeruli without GCH (p < 0.05), consistent with tubular obstruction. Antibody staining revealed fibrin clotting in Bowmans space in 4-h samples and this persisted in the 2-d samples. Heparin reduced and aminocaproic acid increased the GCH seen in 4-h samples. Tubular dilation was evident with injury to the epithelium after 2 d. After one week, areas of inflammatory cell infiltration were present. After four weeks, areas of interstitial fibrosis were revealed by Massons trichrome stain. The consequences of GCH induced by diagnostic ultrasound with contrast agents include rupture of glomerular capillaries, procoagulant activity resulting in intratubular obstruction, and the potential for progression of the resulting tubular injury toward interstitial fibrosis.

Contrast-Enhanced Diagnostic Ultrasound Causes Renal Tissue Damage in a Porcine Model

Objective. Glomerular capillary hemorrhage (GCH) has been reported and confirmed as a consequence of contrast‐enhanced diagnostic ultrasound (CEDUS) imaging of rat kidney. This study assessed renal tissue injury in the larger porcine model. Methods. The right kidneys of anesthetized pigs were imaged in 8 groups of 4 pigs. A Vingmed System Five ultrasound machine (General Electric Co, Cincinnati, OH) was used at 1.5 MHz in the B‐mode to intermittently scan the kidney at 4‐second intervals. An Acuson Sequoia 512 machine (Siemens Medical Solutions, Mountain View, CA) was used in the 1.5‐MHz Cadence contrast pulse sequencing mode with intermittent agent clearance bursts at 4‐second intervals. Kidneys were scanned transabdominally or after laparotomy through a saline standoff. The second machines probe was placed in contact with the kidney for 1 group. A perflutren lipid microsphere contrast agent (Definity; Lantheus Medical Imaging, Inc, North Billerica, MA) was infused at 4 μL/kg/min (diluted 33:1 in saline) for 4 minutes during scanning. Results. Blood‐filled urinary tubules were evident on the kidney surface for all groups except the group with the probe in contact with the kidney. Glomerular capillary hemorrhage was found by histologic processing in 31.7% ± 9.8% (mean ± SD) of glomeruli in the center of the scan plane for 1.7‐MPa transabdominal scanning and 1.5% ± 2.9% of glomeruli in sham samples (P < .05). In addition, hematuria was detected after scanning, and tubular obstruction occurred in some nephrons. Conclusions. Renal tissue damage was induced by CEDUS in the porcine model. This result, together with previous studies in rats, support a hypothesis that GCH would occur in humans from similar CEDUS exposure.

Anesthetic Techniques Influence the Induction of Pulmonary Capillary Hemorrhage During Diagnostic Ultrasound Scanning in Rats

Pulmonary capillary hemorrhage can be induced by diagnostic ultrasound (US) during direct pulmonary US scanning in rats. The influence of specific anesthetic techniques on this bioeffect was examined.

Dependence of Thresholds for Pulmonary Capillary Hemorrhage on Diagnostic Ultrasound Frequency

Pulmonary ultrasound examination has become routine for diagnosis in many clinical and point-of-care medical settings. However, the phenomenon of pulmonary capillary hemorrhage (PCH) induction during diagnostic ultrasound imaging presents a poorly understood risk factor. PCH was observed in anesthetized rats exposed to 1.5-, 4.5- and 12.0-MHz diagnostic ultrasound to investigate the frequency dependence of PCH thresholds. PCH was detected in the ultrasound images as growing comet tail artifacts and was assessed using photographs of the surface of excised lungs. Previous photographs acquired after exposure to 7.6-MHz diagnostic ultrasound were included for analysis. In addition, at each frequency we measured dosimetric parameters, including peak rarefactional pressure amplitude and spatial peak, pulse average intensity attenuated by rat chest wall samples. Peak rarefactional pressure amplitude thresholds determined at each frequency, based on the proportion of PCH in groups of five rats, were 1.03 ± 0.02, 1.28 ± 0.14, 1.18 ± 0.12 and 1.36 ± 0.15 MPa at 1.5, 4.5, 7.6 and 12.0 MHz, respectively. Although the PCH lesions decreased in size with increasing ultrasonic frequency, owing to the smaller beam widths and scan lengths, the peak rarefactional pressure amplitude thresholds remained approximately constant. This dependence was different from that of the mechanical index, which indicates a need for a specific dosimetric parameter for safety guidance in pulmonary ultrasound.

In Vivo Gas Body Efficacy for Glomerular Capillary Hemorrhage Induced by Diagnostic Ultrasound in Rats

Glomerular capillary hemorrhage (GCH) in rat kidney provided a model for assessing in vivo gas body efficacy in diagnostic or therapeutic applications of ultrasound. Two diagnostic ultrasound machines were utilized: one monitored the harmonic B-mode contrast enhancement of the left kidney and the other exposed the right kidney for GCH production. Definity contrast agent was infused at 1, 2, 5, or 10 ¿L/(kg·min) and infusion durations were 30, 60, 120, or 300 s. Exposure of the right kidney was at a peak rarefactional pressure amplitude of 2.3 MPa at 1.5 MHz. The circulating dose was estimated with a simple model of agent dilution and gas body loss. For 300 s infusion at 5 ¿L/(kg·min), the left kidney image brightness increased to a plateau with an estimated 6.4 ± 1.3 ¿L/kg circulating dose with no GCH in histological sections. Exposure of the right kidney with a 1-s image interval reduced the estimated circulating dose to 1.3 ± 0.3 ¿ L/kg and induced 68.4% GCH. Dose and duration increases gave rapidly diminishing treatment effectiveness per gas body. The effective in vivo agent dose in rats can be reduced greatly due to high gas body destruction in the small animal, complicating predictions for similar conditions of human treatment.