Glomerular Capillary Hemorrhage Research Articles

The Dependence of Glomerular Capillary Hemorrhage Induced by Contrast Enhanced Diagnostic Ultrasound on Microbubble Diameter

A recently proposed two-criterion model for cavitational bioeffects in tissue with microbubbles (MBs) was tested. The glomerular capillary hemorrhage bioeffect was observed in rat kidney for contrast agent MB suspensions with mean diameters of 1.6, 3.1 and 5.5 µm. A diagnostic ultrasound machine was used at 3.6 MHz and 5.5 MHz for intermittent scans at power settings 2 dB apart. Petechial hemorrhage counts scored on the surface of the kidneys, and glomeruli were scored in histology. Thresholds for the petechial hemorrhage measurements were the same for the large and medium MB suspensions but substantially higher for the small MBs. For the histology, the medium MBs gave a higher threshold than the large MBs at 5.5 MHz. The pressure amplitude thresholds are in approximate agreement with theory, and the optimum MB size counterintuitively increased for increasing ultrasound frequency, as predicted. The two-criterion model of MB-associated capillary hemorrhage is supported.

A Two-Criterion Model for Microvascular Bio-Effects Induced In Vivo by Contrast Microbubbles Exposed to Medical Ultrasound

The mechanical index (MI) is a theoretical exposure parameter for cavitational bio-effects of diagnostic ultrasound. The theory for the MI assumed that bubbles of all relevant sizes exist in tissue, a condition that is approximated for tissues that include a microbubble contrast agent. Therefore, the MI should allow science-based safety guidance for contrast-enhanced diagnostic ultrasound. However, theoretical predictions of bio-effects thresholds based on the MI typically do not concur with the frequency dependence of experimentally measured thresholds for bio-effects. For example, experimental thresholds for glomerular capillary hemorrhage in rats infused with contrast microbubbles increased approximately linearly with frequency, whereas the MI predicted a square root dependence. Here, cavitation thresholds were computed for linear versions of the acoustic pulses used in that study assuming bubbles containing either air, C3F8, or a 1:1 mixture of the two and surrounded by either blood or kidney tissue. Although no single threshold criterion was successful, combining results for one criterion that maximized circumferential stress in the capillary wall with another that ensured an inertial collapse produced thresholds that were consistent with experimental data. This suggests that a contrast-specific safety metric may be achieved following validation of this two-criterion model.

Do Anesthetic Techniques Influence the Threshold for Glomerular Capillary Hemorrhage Induced in Rats by Contrast-Enhanced Diagnostic Ultrasound?

Glomerular capillary hemorrhage can be induced by ultrasonic cavitation during contrast-enhanced diagnostic ultrasound (US) exposure, an important nonthermal US bioeffect. Recent studies of pulmonary US exposure have shown that thresholds for another nonthermal bioeffect of US, pulmonary capillary hemorrhage, is strongly influenced by whether xylazine is included in the specific anesthetic technique. The objective of this study was to determine the influence of xylazine on contrast-enhanced diagnostic US-induced glomerular capillary hemorrhage. In this study, anesthesia with ketamine only was compared to ketamine plus xylazine for induction of glomerular capillary hemorrhage in rats by 1.6-MHz intermittent diagnostic US with a microsphere contrast agent (similar to Definity; Lantheus Medical Imaging, Inc, North Billerica, MA). Glomerular capillary hemorrhage was measured as a percentage of glomeruli with hemorrhage found in histologic sections for groups of rats scanned at different peak rarefactional pressure amplitudes. There was a significant difference between the magnitude of the glomerular capillary hemorrhage between the anesthetics at 2.3 MPa, with 45.6% hemorrhage for ketamine only, increasing to 63.2% hemorrhage for ketamine plus xylazine (P < .001). However, the thresholds for the two anesthetic methods were virtually identical at 1.0 MPa, based on linear regression of the exposure response data. Thresholds for contrast-enhanced diagnostic US-induced injury of the microvasculature appear to be minimally affected by anesthetic methods.

A two-criterion model for microvascular bioeffects induced in vivo by contrast microbubbles exposed to medical ultrasound

The mechanical index (MI) assumes that bubbles of all relevant sizes exist in tissue, yet that assumption is approximated only in studies that include use of a microbubble contrast agent. If the MI is taken to be the key dosimetric parameter, then it should allow science-based safety guidance for contrast-enhanced diagnostic ultrasound. However, theoretical predictions based on the MI typically do not concur with the frequency dependence of experimentally measured thresholds for bioeffects. For example, experimental measurements of thresholds for glomerular capillary hemorrhage in rats infused with contrast microbubbles (Miller et al. UMB 2008;34:1678) increase approximately linearly with frequency while the MI assumes a square-root dependence. Here, thresholds for inertial cavitation were computed for linear versions of the acoustic pulses used in that study assuming bubbles containing either air, C3F8, or a 1:1 mixture of the two and surrounded by either blood or kidney tissue. While no single threshold criterion was successful, combining results for one criterion that maximized circumferential stress in the capillary wall with another that ensured an inertial collapse, produced thresholds that were consistent with experimental data. This suggests that development of a contrast-specific safety metric may be achieved by further testing and confirmation in different tissues.

Renal interstitial permeability changes induced by microbubble-enhanced diagnostic ultrasound

Ultrasound-targeted microbubble (MB) destruction (UTMD) has been shown to increase the glomerular permeability, providing a potential novel therapeutic approach in targeted drug release for kidney diseases. Therefore, we investigated the impact of UTMD on renal interstitial permeability using MB-mediated diagnostic ultrasound (DUS). The left kidney of Sprague–Dawley (SD) rat was insonated by UTMD with either continuous or intermittent mode for 5 min. Evans blue (EB) revealed that both modes induced renal vascular permeability increase after DUS but recovered after 24 h. Intermittent insonation caused more severe injury than continuous mode. Red blood cells leaked out of the capillaries into interstitium without glomerular capillary hemorrhage (GCH) by hematoxylin and eosin (HE) staining. Electronic microscopy revealed the disruption of focal capillary wall in interstitial tissues. Morphological results confirmed capillary wall recovered in 24 h post-treatment. Results from fluorescence-labeled MBs showed that MBs were mainly localized in the interstitial portion of the tubular region and retained at 24 h. Intriguingly, urinalysis showed no clinical proteinuria after treatment. Our results indicated that MB plus DUS specifically and reversibly enhanced the interstitial permeability without affecting glomerulus, which may be developed into a therapeutic approach for targeting drug release to individual renal compartments.

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

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. 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 machine's 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. 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. 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.

In VivoGas 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 microL/(kg x 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 microL/(kg x min), the left kidney image brightness increased to a plateau with an estimated 6.4 +/- 1.3 microL/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 microL/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.

Gas body destruction reduces the effective circulating dose of ultrasound contrast agent infused in rats.

Glomerular capillary hemorrhage (GCH) in rat kidney provides a model system for assessing in vivo gas body efficacy in diagnostic or therapeutic applications of ultrasound. Two diagnostic ultrasound machines were utilized: one monitored the second‐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 5 μl/kg/min for 300 s during shams and 1.5‐MHz intermittent exposures at 2.3‐MPa peak rarefactional pressure amplitude in groups of five rats. The left kidney image brightness enhancement, indicative of circulating gas body dose, was 18.4 au (decompressed arbitrary units) in shams with no GCH in histology. Exposure of the right kidney with a normal 1‐s image interval induced 68.4% GCH but reduced the left kidney enhancement to 3.3 au, which implies substantial gas body destruction. Decreased exposure with 10‐s interval reduced right kidney GCH (P′<0.001) but only to 30.3% while ameliorating gas body destruction with 13.1‐au left kidney enhancement. The effective in vivo gas body dose in rats may be reduced greatly due to gas body destruction in the small animal, complicating extrapolation to similar conditions of human exposure. [Work supported by NIH grant EB00338.]

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 μl/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 Bowman's 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 Bowman's 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 Masson's 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. (E-mail: douglm@umich.edu)

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. (E-mail: douglm@umich.edu)

Frequency dependence of kidney injury induced by contrast‐aided diagnostic ultrasound

Glomerular capillary hemorrhage (GCH) induced in rat kidneys by contrast aided diagnostic ultrasound was examined as a function of ultrasonic frequency. The right kidneys of anesthetized rats mounted in a water bath were exposed to image pulse sequences at 1 s intervals during intravenous infusion of diluted ultrasound contrast agent. Diagnostic ultrasound (DUS) scanners were utilized for exposure at 1.5, 2.5, 3.2, 5.0 and 7.4 MHz. A laboratory exposure system (LES) was used to simulate DUS exposure at 1.0, 1.5, 2.25, 3.5, 5.0 and 7.5 MHz at relatively high peak rarefactional pressure amplitudes (PRPAs). The exposed kidneys were removed and fixed for histology. GCH was measured by counting glomeruli with blood in the urinary (Bowman's) space on histological sections. The percentage of GCH at a fixed exposure frequency showed a rapid rise with PRPA above an apparent threshold. The threshold values were approximately proportional to the ultrasound frequency, with proportionality constants of 0.5 MPa/MHz for DUS and 0.6 MPa/MHz for LES 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.

Simulation of diagnostic ultrasound image pulse sequences in cavitation bioeffects research

Research on cavitational bioeffects of diagnostic ultrasound (DUS) typically involves a diagnostic scanner as the exposure source. However, this can limit the ranges of exposure parameters for experimentation. Anesthetized hairless rats were mounted in a water bath and their right kidneys were exposed to ultrasound. Amplitude modulation with Gaussian envelopes simulated the image pulse sequences (IPSs) produced by diagnostic scanning. A 10 mulkgmin IV dose of Definity((R)) contrast agent was given during 1-5 min exposures. Glomerular capillary hemorrhage was assessed by histology. A stationary exposure approximated the bioeffects induced by DUS within the beam area. However, the use of five closely spaced exposures more faithfully reproduced the total effect produced within a DUS scan plane. Single pulses delivered at 1 s intervals induced the same effect as the simulated DUS. Use of 100 ms triangle-wave modulations for ramp-up or ramp-down of the IPS gave no effect or a large effect, respectively. Finally, an air-backed transducer simulating DUS without contrast agent showed a zero effect even operating at twice the present DUS guideline upper limit. Relatively simple single-element laboratory exposure systems can simulate diagnostic ultrasound exposure and allow exploration of parameter ranges beyond those available on present clinical systems.

Doppler Mode Pulse Sequences Mitigate Glomerular Capillary Hemorrhage in Contrast-Aided Diagnostic Ultrasound of Rat Kidney

Glomerular capillary hemorrhage (GCH) induced in rat kidney by diagnostic ultrasound involving contrast agent destruction was characterized for different modes to explore possible mitigation strategies. Anesthetized hairless rats were scanned at 2.5 MHz in a water bath with contrast agent infused at 10 microl/kg/minute via tail vein. B mode flash echo imaging (FEI), color Doppler (CD) FEI and realtime Doppler imaging at 1 frame per second were tested, which had image pulse sequences of approximately 0.53 ms, 15.8 ms, and 83.5 ms duration, respectively. Bioeffects endpoints included grossly observed blood-filled tubules, histological evaluation of GCH, and detection of hematuria. B mode FEI for 1 minute induced GCH in 38.6+/-17.1% of glomeruli in histology from the scan plane for a peak rarefactional pressure amplitude (RPA) of 2.6 MPa. The threshold for GCH was approximately 1.5 MPa, confirmed by 10-minute exposure with agent infusion. Paradoxically, CD mode FEI delivered many more pulses but produced less GCH (P < 0.02), and real-time Doppler mode induced only 5.3 +/- 3.8% (P < 0.005). Hematuria results followed the GCH trends. These findings indicate a promising strategy, which is to use relatively slow ramp-up of pulse RPAs in agent-destroying image pulse sequences, for mitigating potential bioeffects in contrastaided diagnostic ultrasound.