Christina P. Keravnou

DYNAMIC CONTRAST-ENHANCED ULTRASOUND OF SLAUGHTERHOUSE PORCINE LIVERS IN MACHINE PERFUSION

The aim of this study was to enable investigations into novel imaging and surgical techniques by developing a readily accessible, versatile liver machine perfusion system. Slaughterhouse pig livers were used, and dynamic contrast-enhanced ultrasound was introduced to optimize the procurement process and provide real-time perfusion monitoring. The system comprised a single pump, oxygenator, bubble trap and two flowmeters for pressure-controlled perfusion of the vessels using an off-the-shelf perfusate at room temperature. Successful livers exhibited homogeneous perfusion in both the portal vein and hepatic artery with dynamic contrast-enhanced ultrasound, which correlated with stable oxygen uptake, bile production and hepatic resistance and normal histology at the end of 3 h of perfusion. Dynamic contrast-enhanced ultrasound revealed perfusion abnormalities invisible to the naked eye, thereby providing context to the otherwise systemic biochemical/hemodynamic measurements and focal biopsy findings. The model developed here is a simple, cost-effective approach for stable ex vivo whole-organ machine perfusion.

Effects of air embolism size and location on porcine hepatic microcirculation in machine perfusion.

The handling of donor organs frequently introduces air into the microvasculature, but little is known about the extent of the damage caused as a function of the embolism size and distribution. Here we introduced embolisms of different sizes into the portal vein, the hepatic artery, or both during the flushing stage of porcine liver procurement. The outcomes were evaluated during 3 hours of machine perfusion and were compared to the outcomes of livers with no embolisms. Dynamic contrast‐enhanced ultrasound (DCEUS) was used to assess the perfusion quality, and it demonstrated that embolisms tended to flow mostly into the left lobe, occasionally into the right lobe, and rarely into the caudate lobe. Major embolisms could disrupt the flow entirely, whereas minor embolisms resulted in reduced or heterogeneous flow. Embolisms occasionally migrated to different regions of the same lobe and, regardless of their size, caused a general deterioration in the flow over time. Histological damage resulted primarily when both vessels of the liver were compromised, whereas bile production was diminished in livers that had arterial embolisms. Air embolisms produced a dose‐dependent increase in vascular resistance and a decline in oxygen consumption. This is the first article to quantify the impact of air embolisms on microcirculation in an experimental model, and it demonstrates that air embolisms have the capacity to degrade the integrity of donor organs. The extent of organ damage is strongly dependent on the size and distribution of air embolisms. The diagnosis of embolism severity can be safely and easily made with DCEUS. Liver Transpl 20:601‐611, 2014.

Accurate measurement of microbubble response to ultrasound with a diagnostic ultrasound scanner

Ultrasound and microbubbles are often used to enhance drug delivery and the suggested mechanisms are extravasation and sonoporation. Drug delivery schemes with ultrasound and microbubbles at both low and high acoustic amplitudes have been suggested. A diagnostic ultrasound scanner may play a double role as both an imaging and a therapy device. It was not possible to accurately measure microbubble response with an ultrasound scanner for a large range of acoustic pressures and microbubble concentrations until now, mainly because of signal saturation issues. A method for continuously adjusting the receive gain of a scanner and limiting signal saturation was developed to accurately measure backscattered echoes from microbubbles for mechanical indexes (MIs) up to 2.1. The intensity of backscattered echoes from microbubbles increased quarticly with MI without reaching any limit. The signal intensity from microbubbles was found to be linear with concentration at both low and high MIs. However, at very high concentrations, acoustic shadowing occurs which limits the delivered acoustic pressure in deeper areas. The contrastto- tissue ratio was also measured and found to stay constant with MI. These results can be used to better guide drug delivery approaches and to also develop imaging techniques for therapy procedures.

Method for Estimating the Acoustic Pressure in Tissues Using Low-Amplitude Measurements in Water

The aim of this study was to evaluate a simple, reliable and reproducible method for accuracy in estimating the acoustic pressure delivered in tissue exposed to ultrasound. Such a method would be useful for therapeutic applications of ultrasound with microbubbles, for example, sonoporation. The method is based on (i) low-amplitude water measurements that are easily made and do not suffer from non-linear propagation effects, and (ii) the attenuation coefficient of the tissue of interest. The range of validity of the extrapolation method for different attenuation and pressure values was evaluated with a non-linear propagation theoretical model. Depending on the specific tissue attenuation, the method produces good estimates of pressures in excess of 10 MPa. Ex vivo machine-perfused pig liver tissue was used to validate the method for source pressures up to 3.5 MPa. The method can be used to estimate the delivered pressure in vivo in diagnostic and therapeutic applications of ultrasound.

Effects of perfusion and vascular architecture on contrast dispersion: Validation in ex-vivo porcine liver under machine perfusion

Dynamic contrast enhanced ultrasound (DCE-US) enables imaging of cancer angiogenesis by quantification of perfusion and dispersion. Although increased perfusion may be found in areas of active angiogenesis due to increased demands for blood supply, decreased perfusion may be caused by the decreased efficiency and functionality, typical of cancer angiogenic microvasculature. Contrast dispersion, mainly determined by the flow profile in large vessels and by the multipath trajectories in the microvasculature, may thus represent a suitable alternative to characterize cancer angiogenesis. Based on a model of the contrast transport kinetics as a convective-dispersion process, several DCE-US methods have been proposed estimating dispersion for characterization of cancer angiogenic vasculature. Although dispersion imaging has shown promising in a clinical context, its physical link with variations in flow and vascular architecture has never been shown. The objective of this work is thus to investigate the influence of flow and underlying vascular architecture on the estimation of dispersion in an ex-vivo machine-perfused pig liver.

Ultrasound and microbubbles induced extravasation in a machine-perfused pig liver

Ultrasound driven microbubbles interact with capillaries and cells and have been proposed to be beneficial for localized drug delivery and uptake. Some of the mechanisms involved is transient cell membrane permeability alteration and vessel poration which increase particle extravasation. Ex vivo machine perfusion of human-sized organs is a technique that provides an ideal environment for preclinical investigations with high physiological relevance not possible with in vitro experiments. In this work, ex vivo machine-perfused pig livers combined with an image-guided therapy system were used to investigate extravasation induced by ultrasound driven microbubbles. Local microvascular flow changes (measured by contrast enhanced ultrasound) and leakage of Evans blue dye in liver parenchyma were used to assess the degree of extravasation. 1—4 MPa peak negative pressure and 20—1000 cycles were considered. Liver areas that were exposed to long pulses (1000 cycles) and peak negative pressure >2.5 MPa showed a detec...

Parametric array for tissue harmonic imaging

More than 50% of abdominal scans are performed on technically difficult patients due to worldwide increase of obesity. Tissue harmonic imaging has shown some improvements on obese patients but the problem remains unsolved. The use of dual frequency pulses allows the generation of sum and difference frequencies in tissue. We hypothesize that the difference frequency (Δf, parametric array) can survive larger penetration depths and increase SNR while maintaining the spatial characteristics of the primary wave. Our objective was to optimize the parameters for efficient generation of the parametric array and study its spatial characteristics for imaging obese patients. Numerical simulations using KZK equation and measurements in tissue-like media were performed to study parametric arrays. Pulses with two frequencies (f 1, f 2) were considered where 0.1f 1≤Δf≤f 1. Pulse inversion was utilized to isolate even harmonics, including Δf. The amplitude of Δf was maximized at Δf = f 1. The axial resolution is not seri...

Microbubble response to dual frequency excitation for broadband contrast imaging

Contrast imaging relies on pulsing schemes like pulse inversion (PI), power-modulated pulse inversion (PMPI), and power modulation (PM) to isolate certain harmonic components. These multi-pulse schemes normally employ single frequency pulses (SFP). In this work, we propose dual frequency pulses (DFP) coupled with pulsing schemes (PI, PMPI, and PM) for microbubble imaging. The proposed DFP contain an amount b 1 of a fundamental frequency f and an amount b 2 of its phase-shifted (φ) second harmonic 2f. Microbubbles were excited with a variety of DFP combinations (b 1, b 2, φ) and their nonlinear response was compared to that from conventional SFP. All pulsing schemes benefit in terms of overall signal from the use of DFP by ~4–6 dB. PI benefits the most when coupled with DFP as it extracts an additional amount of nonlinear fundamental signal (~25 dB) something not feasible with SFP. It was observed that microbubble nonlinear response changes when the relative amount of f and 2f in DFP change, allowing the d...

Contrast-Enhanced Ultrasound for Thrombus Dissolution in an In Vitro Model of Acute Deep Venous Thrombosis

were used to compare subgroup characteristics as well as to assess for longitudinal change in outcome measures. Results: Sixty-three patients, 27 CDT and 36 USAT, were treated for massive (12.7%) or submassive (87.3%) PE; 96.8% were treated for bilateral PE. Patients in the CDT group were more likely to carry a diagnosis of previous deep venous thrombosis and PE. Baseline demographics, PE subtype, echocardiographic parameters, and biomarkers of cardiac injury such as troponin I and brain natriuretic peptide did not differ between the two treatment groups. There was no difference in total dose of lytic administered (23.2 6 13.7mg, CDT vs 27.5 6 12.9 mg, USAT; P 1⁄4 .2). The CDT group had significantly longer hospital stay (11.4 6 8.1 days vs 6.3 6 4.4 days; P 1⁄4 .002) and trended toward lengthier intensive care unit stays. Two patients in the CDT group and one in the USAT group required conversion to surgical thrombectomy (7.4% vs 2.8%; P 1⁄4 .6). Rates of major and minor bleeding complications (11.0% vs 13.9%), PE-related mortality (0.0% vs 2.8%), and 90-day all-cause mortality (7.4% vs 5.6%) did not differ significantly between the CDT and USAT groups. Mortality rate at 1 year was 13.6% for the total cohort; estimated survival at 1 year by Kaplan-Meier analysis was 86.4% overall, 90.2% for CDT, and 78.8% for USAT, respectively (P 1⁄4 .8). Both groups had significant improvement in oxygen requirement at discharge and follow-up (P < .001), and the number of patients requiring oxygen on discharge did not differ between groups. All echocardiographic parameters improved significantly from baseline to 1-year followup; although there were no significant differences between the two groups, the CDT group showed a trend toward greater percentage improvement in right ventricular dilation and estimated pulmonary artery pressure (Table). Conclusions: This study demonstrates no statistical difference in clinical and hemodynamic outcomes or procedural complication rates between USAT and standard CDT for treatment of acute PE. Prospective studies are needed to determine the comparative and cost effectiveness of different interventions for acute massive and submassive PE.

Image-guided sonoporation in an ex vivo machine perfused porcine liver

Sonoporation is the transient and reversible cell membrane permeability change induced with ultrasound and microbubbles. It allows for the uptake of normally impermeable macromolecules and has been suggested for improving drug delivery. The exact sonoporation mechanism and the optimal ultrasound parameters are still under investigation. Ex vivo machine perfused porcine livers are an excellent platform for investigating the sonoporation parameters and specifically the interaction of ultrasound driven microbubbles with the capillaries. Our objective was to identify the ultrasound parameters that are capable of causing detectable perfusion changes in the sonoporation area. Three types of perfusion changes were considered: large mechanical damage void of perfusion, reduced perfusion due to capillary destruction, and unaltered perfusion.